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[Demonstration of tumor inhibiting properties of a strongly immunostimulating low-molecular weight substance. Comparative studies with ifosfamide on the immuno-labile DS carcinosarcoma. Stimulation of the autoimmune activity for approx. 20 days by BA 1, a N-(2-cyanoethylene)-urea. Novel prophylactic possibilities]. A report is given on the recent discovery of outstanding immunological properties in BA 1 [N-(2-cyanoethylene)-urea] having a (low) molecular mass M = 111.104. Experiments in 214 DS carcinosarcoma bearing Wistar rats have shown that BA 1, at a dosage of only about 12 percent LD50 (150 mg kg) and negligible lethality (1.7 percent), results in a recovery rate of 40 percent without hyperglycemia and, in one test, of 80 percent with hyperglycemia. Under otherwise unchanged conditions the reference substance ifosfamide (IF) -- a further development of cyclophosphamide -- applied without hyperglycemia in its most efficient dosage of 47 percent LD50 (150 mg kg) brought about a recovery rate of 25 percent at a lethality of 18 percent. (Contrary to BA 1, 250-min hyperglycemia caused no further improvement of the recovery rate.) However this comparison is characterized by the fact that both substances exhibit two quite different (complementary) mechanisms of action. Leucocyte counts made after application of the said cancerostatics and dosages have shown a pronounced stimulation with BA 1 and with ifosfamide, the known suppression in the post-therapeutic interval usually found with standard cancerostatics. In combination with the cited plaque test for BA 1, blood pictures then allow conclusions on the immunity status. Since IF can be taken as one of the most efficient cancerostatics--there is no other chemotherapeutic known up to now that has a more significant effect on the DS carcinosarcoma in rats -- these findings are of special importance. Finally, the total amount of leucocytes and lymphocytes as well as their time behaviour was determined from the blood picture of tumour-free rats after i.v. application of BA 1. The thus obtained numerical values clearly show that further research work on the prophylactic use of this substance seems to be necessary and very promising.

Effect of etafenone on total and regional myocardial blood flow. The distribution of blood flow to the subendocardial, medium and subepicardial layers of the left ventricular free wall was studied in anaesthetized dogs under normoxic (A), hypoxic (B) conditions and under pharmacologically induced (etafenone) coronary vasodilation (C). Regional myocardial blood flow was determined by means of the particle distribution method. In normoxia a transmural gradient of flow was observed, with the subendocardial layers receiving a significantly higher flow rate compared with the subepicardial layers. In hypoxia induced vasodilation this transmural gradient of flow was persistent. In contrast a marked redistribution of regional flow was observed under pharmacologically induced vasodilation. The transmural gradient decreased. In contrast to some findings these experiments demonstrate that a considerable vasodilatory capacity exists in all layers of the myocardium and can be utilized by drugs. The differences observed for the intramural distribution pattern of flow under hypoxia and drug induced vasodilation support the hypothesis that this pattern reflects corresponding gradients of regional myocardial metabolism.

Pharmacological properties of new neuroleptic compounds. RMI 61 140, RMI 61 144 and RMI 61 280 are newly synthetized N-[8-R-dibenzo(b,f)oxepin-10-yl]-N'-methyl-piperazine-maleates which show interesting psychopharmacologic effects. This work contains the results of a study performed with these three compounds, in order to demonstrate their neuropsycholeptic activity in comparison with chloropromazine (CPZ) and chlordiazepoxide (CPD). The inhibition of motility observed in mice shows that the compounds reduce the normal spontaneous motility as well as the muscle tone. The central-depressant activity is evidenced by increased barbiturate-induced sleep and a remarkable eyelid ptosis can also be observed. Our compounds do not show any activity on electroshock just as do CPZ and CPD. As to the antipsychotic outline, our compounds show strong reduction of lethality due to amphetamine in grouped mice and a strong antiapomorphine activity. They show also an antiaggressive effect and an inhibitory activity on avoidance behaviour much stronger than CPZ. We have also found extrapyramidal effects, as catalepsy, common to many tranquillizers of the kind of the standards used by us. As for vegetative phenomena, the compounds show hypotensive dose related action ranging from moderate to strong, probably due to an a-receptor inhibition. Adrenolytic activity against lethal doses of adrenaline, antiserotonin and antihistaminic effects, as well as other actions (hypothermia, analgesia, etc.) confirm that RMI 61 140, RMI 61 144 and RMI 61 280 are endowed with pharmacologic properties similar and more potent than those of CPZ. Studies on the metabolism of brain catecholamines show that they are similar to CPZ, although with less effect on dopamine level.

[Studies on the action of an anticholinergic agent in combination with a tranquilizer on gastric juice secretion in man]. A double-blind study with intra-individual comparisons was carried out to investigate the effects of 15 mg of (8r)-3alpha-hydroxy-8-isopropyl-1alphaH-tropanium bromide(+/-)-tropate (Sch 1000), 15 mg Sch 1000 + 10 mg oxazepam, 10 mg oxazepam and placebo with oral administration in randomized sequence on gastric juice volume, amount of acid, concentration and pH values in 12 healthy volunteers. The secretion parameters were measured during a 1-h basal period and a 2-h stimulation period. The gastric juice was obtained in 15 min portions via stomach tube. Stimulation was effected by 1 mug/kg/h pentagastrin via drip infusion. The Friedman test was used for the comparative statistical evaluation, and individual comparisons were carried out by means of the Wilcoxon test (pair-differences rank). The results show that Sch 1000 and Sch 1000 + oxazepam were equal in effect on basal and stimulated secretion volume. As compared with placebo, it was not possible to establish an effect on secretion volume for oxazepam alone. Sch 1000 and Sch 1000 + oxazepam were found to be equipotent in reducing the amount of basal acid, while oxazepam reduced this quantity only during the first 30 min of basal secretion. None of the three active preparations was capable of inhibiting the stimulated acid, although both Sch 1000 preparations produced a clear trend towards lowered mean values. During the basal secretion period, all three test preparations had an inhibiting action on acid concentration, but none of them had a significant effect during the stimulation period. The pH value was savely increased only by Sch 1000 and Sch 1000 + oxazepam, and this even only during the basal period. The results are discussed.

Effect of human erythrocyte stromata on complement activation. Stroma from either normal or PNH-like red cells is capable of inhibiting, to some extent, lysis in the sucrose test and enhancing lysis in the acidified-serum test. The same opposing effects are displayed by the exclusion peaks from Sephadex G-200 obtained from each stroma preparation, suggesting that the same factor could be responsible for both activities. Stromata and peaks also induce lysis of PNH-like cells in unacidified serum, indicating activation of complement through the alternate pathway. This is confirmed by immunoelectrophoretic observation. When serum previously activated through the alternate pathway is used in the sucrose test the amount of lysis is markedly reduced. This would indicate that the classical pathway activation can be controlled by the alternate pathway. The possible clinical significance of these factors in determining the haemolytic crisis in PNH patients is discussed.

The effects of processing of barley-based supplements on rumen pH, rate of digestion of voluntary intake of dried grass in sheep. 1. In one experiment the effect on rumen pH of feeding with restricted amounts of whole or pelleted barley was studied. With whole barley there was little variation in rumen pH associated with feeding time, but with pelleted barley the pH decreased from about 7-0 before feeding to about 5-3, 2--3 h after feeding. 2. The rate of disappearance of dried grass during incubation in the rumens of sheep receiving either whole or pelleted barley was studied in a second experiment. After 24 h incubation only 423 mg/g incubated had disappeared in the rumen of sheep receiving pelleted barley while 625 mg/g incubated had disappeared when it was incubated in the rumen of sheep receiving whole barley. 3. The voluntary intake of dried grass of lambs was studied in a third experiment when they received supplements of either 25 or 50 g whole or pelleted barley/kg live weight 0-75. At the high level, pelleted barley reduced intake of dried grass by 534 g/kg but whole barley reduced it by only 352 g/kg. The digestibility of acid-detergent fibre was reduced more by pelleted barley than by whole barley but there was a tendency for a small increase in digestibility of the barley due to processing. 4. The implications of these findings on supplementation of roughages with cereals are discussed.

Poly(8-aminoguanylic acid): formation of ordered self-structures and interaction with poly(cytidylic acid). Poly(8-aminoguanylic acid) has in neutral solution a novel ordered structure of high stability. The 8-amino group permits formation of three hydrogen bonds between two residues along the \"top\", or long axis, of the purines. The usual hydrogen bonding protons and Watson-Crick pairing sites are not involved in the association. The bonding scheme has a twofold rotation axis and is hemiprotonated at N(7). Poly(8NH2G) is converted by alkaline titration (pK = 9.7) to a quite different ordered structure, which is the favored form over the range approximately pH 10-11. The bonding scheme appears to be composed of a planar, tetrameric array of guanine residues, in which the 8-amino group does not participate in interbase hydrogen bonding. Poly (8NH2G) does not interact with poly(C) in neutral solution because of the high stability of the hemiprotonated G-G self-structure. Titration to the alkaline plateau, however, permits ready formation of a two-stranded Watson-Crick helix. In contrast to the monomer 8NH2GMP, poly(8NH2G) does not form a triple helix with poly(C) under any conditions. The properties of the ordered structures are interpreted in terms of a strong tendency of the 8-amino group to form a third interbase hydrogen bond, when this possibility is not prevented by high pH.

Effect of pH on substrate and inhibitor kinetic constants of human liver alanine aminopeptidase. Evidence for two ionizable active center groups. The presence of at least two ionizable active center groups has been detected by a study of the effect of pH upon catalysis of hydrolysis of L-alanyl-beta-naphthylamide by human liver alanine aminopeptidase and upon the inhibition of hydrolysis by inhibitors and substrate analogs. Octanoic acid, octylamine, and peptide inhibitors have been found to be competitive inhibitors and are therefore thought to bind the active center. L-Phe was previously shown to bind the active center since it was found to be a competitive inhibitor of the hydrolysis of tripeptide substrates (Garner, C. W., and Behal, F. J. (1975), Biochemistry 14, 3208). A plot of pKm vs. pH for the substrate L-Ala-beta-naphthylamide showed that binding decreased below pH 5.9 and above 7.5, the points at which the theoretical curve undergoes an integral change in slope. These points are interpreted as the pKa either of substrate ionizable groups or binding-dependent enzyme active center groups. Similar plots of pKm vs. pH for L-alanyl-p-nitroanilide (as substrate) and pKi vs. pH for L-Leu-L-Leu-L-Leu and D-Leu-L-Tyr (as inhibitors) gave pairs fo pKa values of 5.8 and 7.4, 6.0 and 7.5, and 5.7 and 7.5, respectively. All the above substrates (and D-Leu-L-Tyr) have pKa values near 7.5; therefore, the binding-dependent group with a pKa value near 7.5 is possibly this substrate group. Similar plots of pKi vs. pH for the inhibitors L-Phe, L-Met, L-Leu, octylamine, and octanoic acid had only one bending point at 7.7, 7.6, 7.4, 6.3, and 5.9, respectively. Amino acid inhibitors, octylamine, and octanoic acid have no groups with pKa values between 5 and 9. These data indicate that there are two active center ionizable groups with pKa values of approximately 6.0 and 7.5 which are involved in substrate binding or inhibitory amino acid binding but not in catalysis since Vmax was constant at all pH values tested.

Formation of transient complexes in the glutamate dehydrogenase catalyzed reaction. The reaction of glutamate dehydrogenase and glutamate (gl) with NAD+ and NADP+ has been studied with stopped-flow techniques. The enzyme was in all experiments present in excess of the coenzyme. The results indicate that the ternary complex (E-NAD(P)H-kg) is present as an intermediate in the formation of the stable complex (E-NAD(P)H-gl). The identification of the complexes is based on their absorption spectra. The binding of the coenzyme to (E-gl) is the rate-limiting step in the formation of (E-NAD(P)H-kg) while the dissociation of alpha-ketoglutarate (kg) from this complex is the rate-limiting step in the formation of (E-NAD(P)H-gl). The Km for glutamate was 20-25 mM in the first reaction and 3 mM in the formation of the stable complex. The Km values were independent of the coenzyme. The reaction rates with NAD+ were approximately 50% greater than those with NADP+. Furthermore, high glutamate concentration inhibited the formation of (E-NADH-kg) while no substrate inhibition was found with NADP+ as coenzyme. ADP enhanced while GTP reduced the rate of (E-NAD(P)H-gl) formation. The rate of formation of (E-NAD(P)H-kg) was inhibited by ADP, while it increased at high glutamate concentration when small amounts of GTP were added. The results show that the higher activity found with NAD+ compared to NADP+ under steady-state assay conditions do not necessarily involve binding of NAD+ to the ADP activating site of the enzyme. Moreover, the substrate inhibition found at high glutamate concentration under steady-state assay condition is not due to the formation of (E-NAD(P)H-gl) as this complex is formed with Km of 3 mM glutamate, and the substrate inhibition is only significant at 20-30 times this concentration.

Stabilization of the globular structure of ferricytochrome c by chloride in acidic solvents. Increasing concentrations of chloride were found to increase the resolution between two visible absorbance spectral transitions associated with acidification of ferricytochrome c. Analysis of a variety of spectral and viscosity measurements indicates that protonation of a single group having an apparent pK of 2.1 +/- 0.2 and an intrinsic pK of about 5.3 displaces the methionine ligand without significantly perturbing the native globular conformation. Analysis of methylated ferricytochrome c suggests that protonation of a carboxylate ion, most likely a heme propionate residue, is responsible for displacement of the methionine ligand. Addition of a proton to a second group having an apparent pK of 1.2 +/- 0.1 displaces the histidine ligand and unfolds the protein from a globular conformation into a random coil. It is most likely that the second protonation occurs on the imidazole ring of the histidine ligand itself. Chloride is proposed to perturb these transitions by ligation in the fifth coordination position of the heme ion. Such ligation stabilizes a globular conformation of ferricytochrome c at pH 0.0 and 25 degrees.

A competitive labeling method for the determination of the chemical properties of solitary functional groups in proteins. The properties of the functional groups in a protein can be used as built-in-probes of the structure of the protein. We have developed a general procedure whereby the ionization constant and chemical reactivity of solitary functional groups in proteins may be determined. The method may be applied to the side chain of histidine, tyrosine, lysine, and cysteine, as well as to the amino terminus of the protein. The method, which is an extension of the competitive labeling technique using [3H]- and [14C]1-fluoro-2,4-dinitrobenzene (N2ph-F) in a double-labeling procedure, is rapid and sensitive. Advantage is taken of the fact that after acid hydrolysis of a dinitrophenylated protein, a derivative is obtained which must be derived from a unique position in the protein. The method has been applied to the solitary histidine residue of lysozyme, alpha-lytic protease, and Streptomyces griseus (S.G.) trypsin, as well as to the amino terminus of the latter protein. The following parameters were obtained for reaction with N2ph-F at 20 degrees C in 0.1 N KCl: the histidine of hen egg-white lysozyme, pKa of 6.4 and second-order velocity constant of 0.188 M-1 min-1; the histidine of alpha-lytic protease, pKa of 6.5 and second-order velocity constant of 0.0235 M-1 min-1; the histidine of S.G. trypsin, pKa of 6.5 and second-order velocity constant of 0.0328 M-1 min-1; the valyl amino terminus of S.G. trypsin, pKa of 8.1 and second-order velocity constant of 0.403 M-1 min-1. In addition, the results obtained provide clues as to the microenvironments of these functional groups, and indicate that the proteins studied undergo pH-dependent conformational changes which affect the microenvironment, and hence the chemical reactivity of these groups.

Modification of arginine and lysine in proteins with 2,4-pentanedione. Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.

The origin of the alkaline inactivation of pepsinogen. Above pH 8.5, pepsinogen is converted into a form which cannot be activated to pepsin on exposure to low pH. Intermediate exposure to neutral pH, however, returns the protein to a form which can be activated. Evidence is presented for a reversible, small conformational change in the molecule, distinct from the unfolding of the protein. At the same time, the molecule is converted to a form of limited solubility, which is precipitated at low pH, where activation is normally seen. The results are interpreted in terms of the peculiar structure of the pepsinogen molecule. Titration of the basic NH2-terminal region produced an open form, which can return to the native form at neutral pH, but which is maintained at low pH by neutralization of carboxylate groups in the pepsin portion.

Bovine liver dihydrofolate reductase: purification and properties of the enzyme. A purification procedure is reported for obtaining bovine liver dihydrofolate reductase in high yield and amounts of 100-200 mg. A key step in the procedure is the use of an affinity gel prepared by coupling pteroyl-L-lysine to Sepharose. The purified reductase has a specific activity of about 100 units/mg and is homogeneous as judged by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and titration with methotrexate. The products of the first step of Edman degradation indicated a minimum purity of 79%. The reductase has a molecular weight of about 21500 on the basis of amino acid composition and 22100 +/- 300 from equilibrium sedimentation. It is not inhibited by antiserum to the Streptococcus faecium reductase (isoenzyme 2). Unlike the reductase of many other vertebrate tissues, the bovine enzyme is inhibited by mercurials rather than activated and it has a single pH optimum at both low and high ionic strength. However, the position of the pH optimum is shifted and the activity increased by increasing ionic strength. Automatic Edman degradation has been used to determine 34 of the amino-terminal 37 amino acid residues. Considerable homology exists between this region and the corresponding regions of the reductase from S. faecium and from Escherichia coli. This strengthens the idea that this region contributes to the structure of the binding site for dihydrofolate.

Purification and properties of Escherichia coli dihydrofolate reductase. Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate. Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation. Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl. Anions neither inhibit nor activate the enzyme.

The influence of pH on the interaction of inhibitors with triosephosphate isomerase and determination of the pKa of the active-site carboxyl group. Ionization effects on the binding of the potential transition state analogues 2-phosphoglycolate and 2-phosphoglycolohydroxamate appear to be attributable to the changing state of ionization of the ligands themselves, therefore it is unnecessary to postulate the additional involvement of an ionizing residue at the active site of triosephosphate isomerase to explain the influence of changing pH on Ki in the neutral range. The binding of the competitive inhibitor inorganic sulfate is insensitive to changing pH in the neutral range. 3-Chloroacetol sulfate, synthesized as an active-site-specific reagent for triosephosphate isomerase, is used to provide an indication of the pKa of the essential carboxyl group of this enzyme. Previously described active-site-specific reagents for the isomerase were phosphate esters, and their changing state of ionization (accompanied by possible changes in their affinity for the active site) may have complicated earlier attempts to determine the pKa of the essential carboxyl group from the pH dependence of the rate of inactivation. Being a strong monoprotic acid, chloroacetol sulfate is better suited to the determination of the pKa of the carboxyl group. Chloroacetol sulfate inactivates triosephosphate isomerase by the selective esterification of the same carboxyl group as that which is esterified by the phosphate esters described earlier. From the pH dependence of the rate of inactivation of yeast triosephosphate isomerase, the apparent pKa of the active-site carboxyl group is estimated as 3.9 +/- 0.1.

Monoanion inhibition and 35Cl nuclear magnetic resonance studies of renal dipeptidase. Kinetic analyses of monoanion inhibition and 15Cl nuclear magnetic resonance at 5.88 MHz were employed to study monoanion interactions with the zinc metalloenzyme, renal dipeptidase. The enzyme-catalyzed hydrolysis of glycyldehydrophenylalanine exhibited competitive inhibition when the reaction rate was determined in the presence of the monovalent anions fluoride, chloride, bromide, iodide, azide, nitrate, or thiocyanate or upon the addition of the divalent anion, sulfate. Competitive inhibition was produced by these anions. One anion was bound per enzyme molecule, and except in the case of fluoride all of the anions appeared to bind at the same site. Cyanide ion produced a much more effective inhibition of renal dipeptidase than the other monoanions, and it was shown that two cyanide ions were bound per enzyme molecule. An investigation of the effect of pH upon monoanion inhibition suggested that the anion inhibitors bind to the group with a pK of approximately 7.8. Complete dissociation of this group (approximately pH 8.4) eliminates the inhibitory effect of anions. The 35Cl line broadening produced by renal dipeptidase in 0.5 M NaCl solutions was 100 times more effective than that produced by equivalent concentrations of aquozinc(II). The line broadening was dependent upon the concentration of the metalloenzyme and independent of the frequency of the exciting radiation. When zinc ion was removed from the metalloenzyme by dialysis or when chloride was titrated from the metalloenzyme by cyanide, line broadening was decreased. Treatment of renal dipeptidase with saturating concentrations of the competitive inhibitor, guanosine triphosphate, in the presence of 0.5 M NaCl also produced a significant decrease in the 35Cl line width. The 35Cl line broadening produced by renal dipeptidase was shown to decrease with increasing pH through the range pH 5.8-10.8. This line-width variation with pH appeared to result from the titration of a site on the metalloprotein with an approximate pK of 7.4. Temperature studies of 35Cl line broadening by the metalloenzyme in the presence of chloride and cyanide inhibitors suggest that the fast exchange process pertains and that the dominant relaxation mechanism is quadrupolar in nature.

The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. Bovine erythrocyte superoxide dismutase was slowly and irreversibly inactivated by hydrogen peroxide. The rate of this inactivation was directly dependent upon the concentrations of both H2O2 and of enzyme, and its second-order rate constant at pH 10.0 and 25 degrees was 6.7 M-1 sec-1. Inactivation was preceded by a bleaching due to rapid reduction of Cu2+ on the enzyme, and following this there was a gradual reappearance of a new absorption in the visible region, which was coincident with the loss of catalytic activity. Inactivation of the enzyme was pH-dependent and indicated an essential ionization whose pKa was approximately 10.2. Replacement of H2O by D2O raised this pKa but did not diminish the catalytic activity of superoxide dismutase, measured at pH 10.0. Several compounds, including xanthine, urate, formate, and azide, protected the enzyme against inactivation by H2O2. Alcohols and benzoate, which scavenge hydroxyl radical, did not protect. Compounds with special affinity for singlet oxygen were similarly ineffective. The data were interpreted in terms of the reduction of the enzyme-bound Cu2+ to Cu+, by H2O2, followed by a Fenton's type reaction of the Cu+ with additional H2O2. This would generate Cu2+-OH- or its ionized equivalent, Cu2+-O--, which could then oxidatively attack an adjacent histidine and thus inactivate the enzyme. Compounds which protected the enzyme could have done so by reacting with the bound oxidant, in competition with the adjacent histidine.

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide. The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.

Conformational changes induced in a homogeneous anti-type III pneumococcal antibody by oligosaccharides of increasing size. The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.

Evidence of the involvement of a 50S ribosomal protein in several active sites. The functional role of the Bacillus stearothermophilus 50S ribosomal protein B-L3 (probably homologous to the Escherichia coli protein L2) was examined by chemical modification. The complex [B-L3-23S RNA] was photooxidized in the presence of rose bengal and the modified protein incorporated by reconstitution into 50S ribosomal subunits containing all other unmodified components. Particles containing photooxidized B-L3 are defective in several functional assays, including (1) poly(U)-directed poly(Phe) synthesis, (2) peptidyltransferase activity, (3) ability to associate with a [30S-poly(U)-Phe-tRNA] complex, and (4) binding of elongation factor G and GTP. The rates of loss of the partial functional activities during photooxidation of B-L3 indicate that at least two independent inactivating events are occurring, a faster one, involving oxidation of one or more histidine residues, affecting peptidyltransferase and subunit association activities and a slower one affecting EF-G binding. Therefore the protein B-L3 has one or more histidine residues which are essential for peptidyltransferase and subunit association, and another residue which is essential for EF-G-GTP binding. B-L3 may be the ribosomal peptidyltransferase protein, or a part of the active site, and may contribute functional groups to the other active sites as well.

The interaction of phospholipase A2 with micellar interfaces. The role of the N-terminal region. The localization of the previously postulated interface recognition site (IRS) in porcine pancreatic phospholipase A2, required for a specific interaction between the enzyme and organized lipid-water interfaces, was investigated by ultraviolet difference spectroscopy, by measurements of the intrinsic fluorescence of the unique Trp residue, and by protection experiments against specific tryptic hydrolysis. Using the enzymically nondegradable substrate analogues: CnH(2n+1)(0-)OOCH2CH2N+(CH3)3-(H,OH), it is shown that the rather hydrophobic N-terminal sequence of the enzyme, viz., Ala-Leu-Trp-Gln-Phe-Arg, is directly involved in the interaction with the lipid-water interface. Besides hydrophobic probably also polar interactions contribute to the binding process. At neutral or acidic pH the presence of a salt bridge between the N-terminal alpha-NH3+ group and a negatively charged side chain stablizes the interface recognition site and allows the enzyme to penetrate micellar surfaces, even in the absence of metal ion. At alkaline pH, interaction of the enzyme with micellar interfaces requires the presence of Ca2+ (Ba2+) ions.

Phospholipase A2 as a probe of phospholipid distribution in erythrocyte membranes. Factors influencing the apparent specificity of the reaction. The action of snake venom phospholipases A2 in intact human erythrocytes was investigated in detail. The basis phospholipase from Agkistrodon halys blomhifii was found to induce both hydrolysis of membrane phospholipids and total cell hemolysis under certain experimental conditions. The hydrolytic action of the basic enzyme was found to consist of two sequential events: (a) hydrolysis of 70% of the total cell ph osphatidylcholine without any evident hemolysis; and (b) complete hydrolysis of the remaining phosphatidylcholine, followed closely by extensive phosphatidylethanolamine hydrolysis and finally with onset of hemolysis, attack on the phosphatidylserine. At pH 7.4 and 10 mM Ca2+ only stage (a) occurred. However, a slight elevation of the pH of incubation to pH 8.0 and/or inclusion of 40 mM Ca2+ in the reaction mixture caused both events (a) and (b) to occur. The addition of glucose limited the action of the enzyme to stage (a) under any reaction conditions. An investigation showed that enzymically induced hemolysis occurred under conditions where the intracellular ATP levels were lowered. Data are presented which suggest that stage (b) is mediated by in influx of Ca2+ into the cell when the levels of ATP are low. Interestingly the phosphllipase from Naja naja venom (Pakistan) yielded results similar to those observed with the basic enzyme from Agkistrodon venom. However, the enzyme from Crotalus adamanteus and the acidic enzyme also present in the Agkistrodon venom produced only slight hydrolysis or hemolysis under any of the conditions studied. Other species of erythrocytes, e.g., guinea pig, monkey, pig, and rat, were tested but only those from guinea pig behaved similarly to the human cells. Pig, monkey, and rat erythrocytes underwent very limited hydrolysis and hemolysis. It is evident that the use of these phospholipases to probe the localization of phospholipds in erythrocyte membranes must be approached with caution. Certain facets of this problem are discussed.

Subunit interactions in yeast glyceraldehyde-3-phosphate dehydrogenase. The spontaneous inactivation of yeast glyceraldehyde-3-phosphate dehydrogenase was found to fit a simple two-state model at pH 8.5 and 25 degrees. The first step is a relatively rapid dissociation of the tetramer to dimers with the equilibrium largely in favor of the tetramer. In the absence of NAD+ the dimer inactivates irreversibly. The apoenzyme is quite stable with a half-life for complete activity loss proportional to the square root of the enzyme concentration. Perturbances of the protein structure (by pH, ionic strength, and specific salts), which have no effect on the tetrameric state of the molecule, result in an alteration of the cooperativity of NAD+ binding, the reactivity of the active-site sulfhydryl group, and the catalytic activity of the enzyme. Covalent modification of two of the four active-site sulfhydryl groups has profound effects on the enzymic activity which are mediated by changes in the subunit interactions. Sedimentation analysis and hybridization studies indicate that the interaction between subunits remains strong after covalent modification. Under normal physiological and equilibrium dialysis conditions the protein is a tetramer. Equilibrium dialysis studies of NAD+ binding to the enzyme at pH 8.5 and 25 degrees reveal a mixed cooperativity pattern. A model consistent with these observations and the observed half-of-the-sites reactivity is that of ligand induced sequential conformational changes which are transferred across strongly interacting subunit domains. Methods for distinguishing negatively cooperative binding patterns from mixtures of denatured enzyme and multiple species are discussed.

Kinetic light scattering studies on the dissociation of hemoglobin from Lumbricus terrestris. The kinetics of the pH-induced dissociation of the 3 X 10(6) mol wt hemoglobin from Lumbricus terrestris (the earthworm) have been studied in a light-scattering stopped-flow apparatus. The ligand dependent dissociation data were fit well by a simple sequential model. The data for CO and oxyhemoglobin are consistent with Hb12 leads to 2Hb6 leads to 12Hb. Methemoglobin at pH 7 appears to be hexameric and the dissociation is consistent with the model: Hb6 leads to 6Hb. In a sequential decay scheme for which light-scattering changes are monitored, the relative amounts of rapid and slow phase are determined by the rate constants as well as the molecular weights of intermediate species. Assignment of the hexameric intermediate is supported by an investigation of the sensitivity of the theoretical kinetic curves to the molecular weights of the intermediates. This assignment is further supported by the following: (1) the same model will fit the data for oxy- and CO-hemoglobin at all three temperatures (a 24-29-fold variation in rate constants), (2) evidence from electron microscopy shows hexameric forms, and (3) methemoglobin is apparently stable as a hexamer at pH 7. When CO replaces O2 as the ligand, the dissociation rate increases by a factor of four. The met is about 20 times faster than the initial oxyhemoglobin dissociation rate, but perhaps more relevant for comparing dissociation of the hexamer, the met rate was respectively 100 times and 500 times faster than that for the assumed hexameric forms of CO- and oxy-hemoglobin. The activation energies for the dodecamer to hexamer dissociation and for the dissociation of the hexamer to smaller forms were about 30 kcal/mol for oxy-, CO-, and methemoglobin.

The reversible reduction of horse metmyoglobin by the iron(II) complex of trans-1,2-diaminocyclohexane-N,N,N,n-tetraacetate. The reduction of metmyoglobin by the iron(II) complex of trans-1,2-diaminocyclohexane-N,N,N'N'-tetraacetate (FeCDTA2-) has been investigated. The equilibrium constant, measured spectrophotometrically, is 0.21 with a resulting reduction potential of 0.050 V for Mb0. The rate constant for the reduction is 28 M-1 sec-1 with a deltaH ++ of 13 kcal M-1 and deltaS ++ of -11 eu. Both CN- and OH- inhibit the reduction because of the relatively low reactivity of cyanometmyoglobin (Mb+CN-) and ionized metmyglobin (Mb+OH-). The rate constant for the reduction of Mb+CN- by FeCDTA2- is 4.0 X 10(-2) M-1 sec-1 and that for reduction of Mb+OH- is 4.8 M-1 sec-1. The nitric oxide complex of metmyoglobin is reduced with a rate constant of 10 M-1 sec-1. The kinetics of oxidation of oxymyoglobin by FeCDTA- were studied. The data are consistent with a mechanism where oxidation takes place entirely through the deoxy form. A rate constant of 1.45 X 10(2) M-1 sec-1 was calculated for the oxidation of deoxymyoglobin by FeCDTA-, in equilibrium constant and rate constant for reduction. The above data are discussed in terms of a simple outer-sphere reduction reaction.

Constitution and properties of axonal membranes of crustacean nerves. The purification of axonal membranes of crustaceans was followed by measuring enrichment in [3H]tetrodotoxin binding capacity and in Na+, K+-ATPase activity. A characteristic of these membranes is their high content of lipids and their low content of protein as compared to other types of plasmatic membranes. The axonal membrane contains myosin-like, actin-like, tropomyosin-like, and tubulin-like proteins. It also contains Na+, K+-ATPase and acetylcholinesterase. The molecular weights of these two enzymes after solubilization are 280,000 and 270,000, respectively. The molecular weights of the catalytic subunits are 96,000 for ATPase and 71,000 for acetylcholinesterase. We confirmed the presence of a nicotine binding component in the axonal membrane of the lobster but we have been unable to find [3H]nicotine binding to crab axonal membranes. The binding to axonal membranes og of the sodium channel, has been studied in detail. The dissociation constant for the binding of [3H]tetrodotoxin to the axonal membrane receptor is 2.9 nM at pH 7.4. The concentration of the tetrodotoxin receptor in crustacean membranes is about 10 pmol/mg of membrane protein, 7 times less than the acetylcholinesterase, 30 times less than the Na+, K+-ATPase, and 30 times less than the nicotine binding component in the lobster membrane. A reasonable estimate indicates that approximately only one peptide chain in 1000 constitutes the tetrodotoxin binding part of the sodium channel in the axonal membrane. Veratridine, which acts selectively on the resting sodium permeability, binds to the phospholipid part of the axonal membrane. [3H]Veratridine binding to membranes parallels the electrophysiological effect. Veratridine and tetrodotoxin have different receptor sites. Although tetrodotoxin can repolarize the excitable membrane of a giant axon depolarized by veratridine, veratridine does not affect the binding of [3H]tetrodotoxin to purified axonal membranes. Similarly, tetrodotoxin does not affect the binding of [3H]veratridine to axonal membranes. Scorpion neurotoxin I, a presynaptic toxin which affects both the Na+ and the K+ channels, does not interfere with the binding of [3H]tetrodotoxin or [3H]veratridine to axonal membranes. Tetrodotoxin, veratridine, and scorpion neurotoxin I, which have in common the perturbation of the normal functioning of the sodium channel, act upon three different types of receptor sites.

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae. 1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.

The reaction between the superoxide anion radical and cytochrome c. 1. The superoxide anion radical (O2-) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O2- and ferrocytochrome c. 2. At 20 degrees C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4-10(6) M-1. S -1 and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O2- and the form of cytochrome c which exists above pH approximately 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O2- reacts with the form present below pH 7.45 with k = 1.4-10(6) M-1 - S-1, the form above pH 7.45 with k = 3.0- 10(5) M-1 - S-1, and the form present above pH 9.2 with k = 0. 3. The reaction has an activation energy of 20 kJ mol-1 and an enthalpy of activation at 25 degrees C of 18 kJ mol-1 both above and below pH 7.45. It is suggested that O2- may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex. 4. No reduction of ferricytochrome c by HO2 radicals could be demonstrated at pH 1.2-6.2 but at pH 5.3, HO2 radicals oxidize ferrocytochrome c with a rate constant of about 5-10(5)-5-10(6) M-1 - S-1.

Identification of the 120 mus phase in the decay of delayed fluorescence in spinach chloroplasts and subchloroplast particles as the intrinsic back reaction. The dependence of the level of this phase on the thylakoids internal pH. After a 500 mus laser flash a 120 mus phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Bio-phys. Acta 376, 329-344, indicates that their EPR signal IIf which they suppose to be due to Z+, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 mus phase. We explain our 120 mus phase by the intrinsic back reaction of the excited reaction center in the presence of Z+, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333-342. The redox state of Z+ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the mus region are compared with those obtained in the ms region.

Light-induced changes of absorbance and electron spin resonance in small photosystem II particles. Photosystem II reaction center components have been studied in small system II particles prepared with digitonin. Upon illumination the reduction of the primary acceptor was indicated by absorbance changes due to the reduction of a plastoquinone to the semiquinone anion and by a small blue shifts of absorption bands near 545 nm (C550) and 685 nm. The semiquinone to chlorophyll ratio was between 1/20 and 1/70 in various preparations. The terminal electron donor in this reaction did not cause large absorbance changes but its oxidized form was revealed by a hitherto unknown electron spin resonance (ESR) signal, which had some properties of the well-known signal II but a linewidth and g-value much nearer to those of signal I. Upon darkening absorbance and ESR changes decayed together in a cyclic or back reaction which was stimulated by 3-(3,4 dichlorophenyl)-1,1-dimethylurea. The donor could be oxidized by ferricyanide in the dark. Illumination in the presence of ferricyanide induced absorbance and ESR changes, rapidly reversed upon darkening, which may be ascribed to the oxidation of a chlorophyll a dimer, possibly the primary electron donor of photosystem II. In addition an ESR signal with 15 to 20 gauss linewidth and a slower dark decay was observed, which may have been caused by a secondary donor.

Enzymic reactions of fatty acid hydroperoxides in extracts of potato tuber. II. Conversion of 9- and 13-hydroperoxy-octadecadienoic acids to monohydroxydienoic acid, epoxyhydroxy- and trihydroxymonoenoic acid derivatives. 1. Crude extracts and partially purified enzyme preparations from potato tubers catalyse, at pH 5-7, the conversion of linoleic acid hydroperoxides to a range of oxygenated fatty acid derivatives. 2. 9-D- and 13-L-hydroperoxide isomers are converted at similar rates to equivalent (isomeric) products. 3. The major products from the 13-hydroperoxide isomer were identified as the corresponding monohydroxydienoic acid derivative, threo-11-hydroxy-trans12,13-epoxy-octadec-cis9-enoic acid and 9,12,13-trihydroxy-octadec-trans10-enoic acid. The corresponding products from the 9-hydroperoxide were the monohydroxydienoic acid, 9,10-epoxy-11-hydroxy-octadec-12-enoic acid and 9,10,13-trihydroxy-octadec-11-enoic acid. 4. No separation of activities forming the different products was achieved by partial purification of enzyme extracts. 5. Product formation was unaffected by EDTA, CN-, sulphydryl reagents or glutathione but was reduced by boiling the extracts. 6. This system is compared with the 9-hydroperoxide-specific enzymic formation of divinyl ether derivatives by potato extracts.

Partial purification and properties of microsomal phosphatidate phosphohydrolase from rat liver. Microsomal phosphatidate phosphohydrolase (phosphatidate phosphatase EC 3.1.3.4) was solubilized and fractionated to yield at least two distinct enzymatically active fractions. One, denoted FA, was non-specific, had a relatively high Km for phosphatidic acid and was insensitive to inhibition by diacylglycerol. The second fraction, FB, was specific for phosphatidates, had a low Km, and was inhibited, non-competitively, by diacylglycerol. FA exhibited a sigmoid substrate-activity curve. The isolated FB aggregated to particles of about 10(6) in the absence of salts and could be dissociated by the addition of monovalent cations at ionic strength 0.4-0.6 to about 2-10(5) daltons and thereby doubled its activity. Dissociation was time- and temperature-dependent. F- was inhibitory. Divalent ions were not required for the activity of FA or FB and inhibited at concentrations exceeding 1 mM.

Bile acids. XLVII. 12alpha-Hydroxylation of precursors of allo bile acids by rabbit liver microsomes. Rabbit liver microsomal preparations fortified with 0.1 mM NADPH effectively promote hydroxylation of [3beta-3H]- or [24-14C]allochenodeoxycholic acid or [5alpha,6alpha-3H2]5alpha-cholestane-3alpha,7alpha-diol to their respective 12alpha-hydroxyl derivatives in yields of about 25 or 65% in 60 min. Minor amounts of other products are formed from the diol. The requirements for activity of rabbit liver microsomal 12alpha-hydroxylase resemble those of rat liver microsomes. Of a number of enzyme inhibitors studied only p-chloromercuribenzoate demonstrated a marked ability to inhibit the reaction with either tritiated substrate. There was no difference in the quantity of product produced from the tritiated acid or the 14C-labeled acid. No clear sex difference was found in activity of the enzyme, nor was an appreciable difference noted in activity of the enzyme between mature and immature animals.

Partial purification and properties of a phenobarbital-induced aldehyde dehydrogenase of rat liver. Properties of the phenobarbital induced cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) have been studied in rat liver. 7-12-Fold higher levels were seen in the cytoplasmic activities after phenobarbital treatment in reactor compared to non-reactor animals with high concentrations of acetaldehyde (18 mM) and propionaldehyde (9 mM). No difference was found with 0.12 mM acetaldehyde, 2 mM glycolaldehyde, 6 mM formaldehyde or 0.5 mM betaine aldehyde. The reactor group also had slightly higher activity in the mitochondrial fraction with the high acetaldehyde and propionaldehyde concentrations. In the microsomal fraction, the activities showed no differences at any substrate concentration. An induced aldehyde dehydrogenase was purified 70-fold by chromatographic techniques. It had different molecular and enzymic properties than the main high-Km enzyme normally present in rat liver cytoplasm. The pI of the induced enzyme was about 7.0 as measured by isoelectric focusing. It was active with several aliphatic and aromatic aldehydes but not with formaldehyde, glycolaldehyde or D-glyceraldehyde. The Km-values for propionaldehyde and acetaldehyde were in the millimolar range. Millimolar concentrations of aromatic aldehydes caused a strong substrate inhibition. The enzyme was inhibited by submicromolar concentrations of disulfiram. Estrone, deoxycorticosterone, progesterone and diethylstilbestrol also affected the enzyme activity.

Cholinesterases from plant tissues. VI. Preliminary characterization of enzymes from Solanum melongena L. and Zea mays L. Enzymes capable of hydrolyzing esters of thiocholine have been assayed in extracts of Solanum melongena L. (eggplant) and Zea Mays L. (corn). The enzymes from both species are inhibited by the anti-cholinesterases neostigmine, physostigmine, and 284c51 and by AMO-1618, a plant growth retardant and they both have pH optima near pH 8.0. The enzyme from eggplant is maximally active at a substrate concentration of 0.15 mM acetylthiocholine and is inhibited at higher substrate concentrations. On the basis of this last property, the magnitude of inhibition by the various inhibitors, and the substrate specificity, we conclude that the enzyme from eggplant, but not that from corn, is a cholinesterase.

Behavior of soluble and immobilized acid phosphatase in hydro-organic media. The hydrolysis of p-nitrophenyl phosphate by wheat germ acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) has been investigated in mixtures of aqueous buffers with acetone, dioxane and acetonitrile. The enzyme was either in free solution or immobilized on a pellicular support which consisted of a porous carbonaceous layer on solid glass beads. The highest enzyme activity was obtained in acetone and acetonitrile mixed with citrate buffer over a wide range of organic solvent concentration. In 50% (v/v) acetone both V and Km of the immobilized enzyme were about half of the values in the neat aqueous buffer, but the Ki for inorganic phosphate was unchanged. In 50% (v/v) mixtures of various solvents and citrate buffers of different pH, the enzymic activity was found to depend on the pH of the aqueous buffer component rather than the pH of the hydro-organic mixture as measured with the glass-calomel electrode. The relatively high rates of p-nitrophenol liberation in the presence of glucose even at high organic solvent concentrations suggest that transphosphorylation is facilitated at low water activity.

Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls. A strain of Bacillus sp (Bacillus R-4) produces a protease and a carbohydrolase both of which have the ability to lyse Rhizopus cell walls. Of the enzymes, the carbohydrolase has been purified to an ultracentrifugally and electrophoretically homogeneous state, and identified as a chitosanase. The enzyme was active on glycol chitosan as well as chitosan. Molecular weight of the purified enzyme was estimated as 31 000 and isoelectric point as pH 8.30. The enzyme was most active at pH 5.6 and at 40 degrees C with either Rhizopus cell wall or glycol chitosan as substrate, and was stable over a range of pH 4.5 to 7.5 at 40 degrees C for 3 h. The activity was lost by sulfhydryl reagents and restored by either reduced glutathione of L-cysteine. An abrupt decrease in viscosity of the reaction mixture suggested an endowise cleavage of chitosan by this enzyme.

Specificity studies on alpha-mannosidases using oligosaccharides from mannosidosis urine as substrates. Oligosaccharides containing terminal non-reducing alpha(1 leads to 2)-, alpha(1 leads to 3)-, and alpha(1 leads to 6)-linked mannose residues, isolated from human and bovine mannosidosis urines were used as substrates to test the specificities of acidic alpha-mannosidases isolated from human and bovine liver. The enzymes released all the alpha-linked mannose residues from each oligosaccharide and were most effective on the smallest substrate. Enzyme A in each case was less active on the oligosaccharides than alpha-mannosidase B2, even though the apparent Km value for the substrates was the same with each enzyme. The human acidic alpha-mannosidases were also found to be more active on substrates isolated from human rather than bovine mannosidosis urine. Human alpha-mannosidase C, which has a neutral pH optimum when assayed with a synthetic substrate, did not hydrolyse any of the oligosaccharides at neutral pH, but was found to be active at an acidic pH.

Calcium-stimulated adenosine triphosphatase in the microsomal fraction of tooth germ from porcine fetus. The characterization and localization of a Ca(2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in the tooth germ of the porcine fetus are reported. This enzyme, a microsome fraction, is preferentially activated by Ca(2+). In the presence of 0.5 mM ATP, maximal enzyme activity is obtained at 0.5--1.0 mM CaCl2. The maximal rate of ATP hydrolysis is approx. 20 mumol per h per mg of protein as the enzyme preparation is used here. At optimal Ca(2+) concentration, the Mg(2+) has an inhibitory effect. The enzyme does not require Na+ or/and K+ for activation by Ca(2+). Other nucleotide triphosphates may serve as the substrate, but V for ATP is the highest. The Km for ATP is 8.85 - 10(-5) M. The optimal pH for Ca(2+) activation of the enzyme lies around 9.2. Well known inhibitors of (Na+ + K+)-ATPase, mitochondria ATPase and Ca(2+)-ATPase in the erthrocyte do not inhibit the enzyme. In the subcellular order the enzyme may be assumed to be localized in the smooth endoplasmic reticulum fraction containing cell and Golgi body membrane fragments and in the tissue order in the enamel organ containing an ameloblast layer, stratum intermedium and stellate reticulum.

Preparation and characterization of an enzymatically active immobilized derivative of myosin. Purified skeletal muscle myosin (EC 3.6.1.3) has been covalently bound to Sepharose 4B by the cyanogen bromide procedure. The resulting complex, Sepharose-Myosin, possesses adenosine triphosphatase activity and is relatively stable for long periods of time. Under optimal binding conditions, approximately 33% of the specific ATPase activity of the bound myosin is retained. Polyacrylamide gel electrophoresis of polypeptides released from denatured Sepharose-Myosin indicates that 85% of the myosin is attached to the agarose beads through the heavy chains and the remainder through the light chains, in agreement with predictions of binding and release based upon either the lysine contents or molecular weights of themyosin subunits. The adenosine triphosphatase of the immobilized myosin has been investigated under conditions of varying pH, ionic strength, and cation concentration. The ATPase profiles of immobilized myosin are quite similar to those for free myosin, however subtle differences are found. The Sepharose-Myosin ATPase is not as sensitive as myosin to alterations in salt concentration and the apparent KM is approximately two-fold higher than that of myosin. These differences are probably due to chemical modification in the region of the attachment site(s) to the agarose beads and hydration and diffusion limitations imposed by the polymeric agarose matrix.

Radioactive labeling and location of specific thiol groups in myosin from fast, slow and cardiac muscles. 1. Based on incorporation of radioactively labeled N-ethylmaleimide, the readily reactive thiol groups of isolated myosin (EC 3.6.1.3) from fast, slow and cardiac muscles could be classified into 3 types. All 3 myosins contain 2 thiol-1, 2 thiol-2 and a variable number of thiol-3 groups per molecule. Both thiol-1 and thiol-2 groups which are essential for functioning of the K+-stimulated ATPase, are located in the heavy chains in all 3 myosin types. 2. The variation in the incorporation pattern of N-ethylmaleimide over the 3 thiol group classes under steady-state conditions of Mg(2+) - ATP hydrolysis allowed different conformations of some reaction intermediates to be characterized. In all 3 types of myosin the hydrolytic cycle of Mg(2+) - ATP was found to be controlled by the same step at 25 degrees C. In all three cases, this rate-limiting step is changed in the same way by lowereing temperature. 3. Using the chemically determined molecular weights for myosin light chains, their stoichiometry was found on the basis of sodium dodecyl sulfate electrophoresis to be 1.2 : 2.1 : 0.8 for light chain-1: light chain-2:light chain-3 per molecule of fast myosin, 2.0 : 1.9 for light chain-1:light chain-2 per molecule of slow myosin and 1.9 : 1.9 for light chain-1:light chain-2 per molecule of cardiac myosin. This qualitative difference in light subunit composition between the fast and the two types of slow myosin is not reflected in the small variations of the characteristics exhibited by the isolated myosins, but rather seems to be connected with their respective myofibrillar ATPase activities.

Yeast glutathione reductase. Studies of the kinetics and stability of the enzyme as a function of pH and salt concentration. 1. The pH dependencies of the apparent Michaelis constant for oxidized glutathione and the apparent turnover number of yeast glutathione reductase (EC 1.6.4.2) have been determined at a fixed concentration of 0.1 mM NADPH in the range pH 4.5--8.0. Between pH 5.5 and 7.6, both of these parameters are relatively constant. The principal effect of low pH on the kinetics of the enzyme-catalyzed reaction is the observation of a pH-dependent substrate inhibition by oxidized glutathione at pH less than or equal 7, which is shown to correlate with the binding of oxidized glutathione to the oxidized form of the enzyme. 2. The catalytic activity of yeast glutathione reductase at pH 5.5 is affected by the sodium acetate buffer concentration. The stability of the oxidized and reduced forms of the enzyme at pH 5.5 and 25 degrees C in the absence of bovine serum albumin was studied as a function of sodium acetate concentration. The results show that activation of the catalytic activity of the enzyme at low sodium acetate concentration correlates with an effect of sodium acetate on a reduced form of the enzyme. In contrast, inhibition of the catalytic activity of the enzyme at high sodium acetate concentration correlates with an effect of sodium acetate on the oxidized form of the enzyme.

Characteristics of the dephosphorylated form of phosphorylase purified from rat liver and measurement of its activity in crude liver preparations. The phosphorylated form of liver glycogen phosphorylase (alpha-1,4-glucan : orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) (phosphorylase a) is active and easily measured while the dephosphorylated form (phosphorylase b), in contrast to the muscle enzyme, has been reported to be essentially inactive even in the presence of AMP. We have purified both forms of phosphorylase from rat liver and studied the characteristics of each. Phosphorylase b activity can be measured with our assay conditions. The phosphorylase b we obtained was stimulated by high concentrations of sulfate, and was a substrate for muscle phosphorylase kinase whereas phosphorylase a was inhibited by sulfate, and was a substrate for liver phosphorylase phosphatase. Substrate binding to phosphorylase b was poor (KM glycogen = 2.5 mM, glucose-1-P = 250 mM) compared to phosphorylase a (KM glycogen = 1.8 mM, KM glucose-1-P = 0.7 mM). Liver phosphorylase b was active in the absence of AMP. However, AMP lowered the KM for glucose-1-P to 80 mM for purified phosphorylase b and to 60 mM for the enzyme in crude extract (Ka = 0.5 mM). Using appropriate substrate, buffer and AMP concentrations, assay conditions have been developed which allow determination of phosphorylase a and 90% of the phosphorylase b activity in liver extracts. Interconversion of the two forms can be demonstrated in vivo (under acute stimulation) and in vitro with little change in total activity. A decrease in total phosphorylase activity has been observed after prolonged starvation and in diabetes.

Multiple forms of casein kinase from rabbit erythrocytes. Two rabbit erythrocyte casein kinases, GTP:casein kinase I and GTP:casein kinase II, have been purified 29 000- and 47 000-fold, respectively. Studies employing sucrose density gradient centrifugation indicate that kinase I has a molecular weight of about 9.5 - 10(5) (25 S) and kinase II about 1.4 - 10(6) (32 S). These enzymes can utilize either ATP or GTP as the phosphoryl donor. Among various protein substrates examined, these kinases catalyze the phosphorylation of casein greater than 50% dephosphorylated phosvitin congruent to 50% dephosphorylated casein greater than phosvitin. Histones, protamine and bovine serum albumin are poor phosphoryl acceptors. Kinetic data indicate that both enzymes are inhibited by high casein substrate concentrations which may be partially relieved by NaCl. Both phosphotransferases require Mg(2+) for activity and are optimally active at pH 9.0. The enzymes have apparent Km values of 2.5 - 10(-5) M for GTP, 2 - 10(-5) M for ATP, and 0.4--0.6 mg/ml for casein. The incorporation of the terminal phosphate of GTP into casein as catalyzed by these enzymes is inhibited to varying degrees by ATP, ITP, ADP, and GDP but not by UTP, CTP, GMP, adenosine 3':5'-cyclic monophosphate, and guanosine 3':5'-cyclic monophosphate. In addition, NaF and 2,3-diphosphoglyceric acid are also found to inhibit the activity of both kinases. The effect of 2,3-diphosphoglycerate is interesting and suggests that this metabolite may regulate the activity of the casein kinases in the red blood cells.

Kinetic studies and effects of anions on creatine phosphokinase from skeletal muscle of rhesus monkey (Macaca mulatta). A purification procedure for creatine kinase (EC 2.7.3.2) from muscle of the monke35--170 muequiv H+/mg protein per min at 30 degrees C and a yield of approx. 0.5 g/kg muscle. Assuming equilibrium kinetics, synergistic binding of substrates at one catalytic site is found for both the forward and back reactions. Kinetic constants for the binding of each substrate to the free enzyme and the enzyme-second substrate complex are determined and are compared with those for the enzyme from other species. Inhibition by small anions is determined in the presence of different combinations of substrates and products. SO4(2-) inhibits by simple competitive inhibition and probably binds at the site of the transferrable phosphoryl group. Inhibition by NO3-, NO2-, SCN- and Cl- is more complex and these ions are suggested to mimic the transferrable phosphoryl group in a planar transition-state complex. These anions stabilize the dead-end complex, enzyme-creatine-MgADP, which lacks the transferable phosphoryl group. The effects of these anions on the dissociation constants of the enzyme-substrate complexes is reported and is in accord with the above hypothesis. The dead-end complex in the absence of anion does not protect the essential thiol group against inhibition by iodoacetamide. Addition of NO3- or Cl- to the dead-end complex or a substrate equilibrium mixture without anion confers protection. The essential thiol group is inhibited by iodoacetamide at a rate which is essentially independent of pH over the normal stability range of the enzyme. Contrary to our previous report this pH independence is not altered by the presence of dead-end complex, creatine plus MgADP, in the presence or absence of anion or in the presence of a substrate equilibrium mixture. It is inferred that the 'essential' thiol group of the monkey enzyme has essentially the same properties as that of the rabbit enzyme. In consequence, the inferences made about the role of this group based on our previous work on the monkey enzyme are no longer valid. The present findings are compatible with the essential thiol group playing a conformational role in the catalytic process.

Studies on rat renal cortical cell kallikrein. I. Separation and measurement. A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained. Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83-108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6-10(-2) to 4.6 - 10(-2) alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase units per 10(8) cells. However, cells incubated in a nutrient medium at 37 degrees C for 3-8 h contained no measurable kallikrein activity, whereas the surrounding medium had kallikrein activity which could be significantly increased by aldosterone and decreased by spironolactone.

Studies on electron transfer between mercury electrode and hemoprotein. The electrochemical behaviour of ferricytochrome c, metmyoglobin and methemoglobin was studied using d.c., a.c. and differential pulse polarography, and controlled potential electrolysis. 1. The three hemoproteins yield d.c. polarographic steps, and peaks in differential pulse polarograms, the height of which is proportional to concentration. The charge transfer is influenced by strong adsorption. 2. The concentration dependence of the a.c. polarograms indicates structural changes in the adsorbed molecules. 3. The reduction products of controlled potential electrolysis of metmyoglobin and methemoglobin have absorption spectra identical with the native control samples. The affinity for oxygen and the cooperativity in hemoglobin are not affected by the reaction at the electrode. 4. The charge transfer proceeds via adsorbed, already reduced, molecules to freely diffusible proteins.

The binding of organic phosphates to human methaemoglobin A. Perturbation of the polymerization of proteins by effectors. Theory is presented relating to the binding of an effector to two states of a protein acceptor coexisting in equilibrium. The problem is treated in terms of the four possible cases which specify relations between numbers of binding sites and intrinsic binding constants relevant to the acceptor states. It is shown that a distinction between these cases may be possible on the basis of the form of a plot of unbound effector concentration versus the constituent equilibrium coefficient which may be calculated from the sedimentation coefficient of the protein constituent. Particularly noteworthy in this respect is the finding that a turning point may exist in this plot for defined conditions with systems in which binding sites are not conserved (and binding affinities are altered) on polymer formation. The latter type of system is exemplified by studies on methaemoglobin A in 0.25 M sodium acetate, pH 5.4. In the absence of added organic phosphate effectors, a dimer-tetramer equilibrium operates governed by an association constant of 4.15 +/- 0.06 X 10(3) 1/mol, determined from sedimentation equilibrium results. Correlation of sedimentation velocity and equilibrium results shows that addition of adenosine 5'-triphosphate (ATP) results in its binding to one site on each of the dimeric (alpha beta) and tetrameric (alpha beta)2 species with intrinsic binding constants 1.03-10(3)-1.20-10(3) and 1.1-10(4)-2.1-10(4) 1/mol, respectively. It is also shown that 2,3-diphosphoglycerate perturbs the dimer-tetramer equilibrium in a similar way to ATP.

N-terminal spin label studies of hemoglobin, Ligand and pH dependence. Human hemoglobin was spin labeled with 4-isothiocanato-2,2,6,6-tetramethyl-piperdinooxyl, which is known to bind specifically to the N-terminal alpha-amino groups of proteins and slightly to the reactive sulfhydryl groups. Electron spin resonance (ESR) analysis indicated a partially resolved five-line spectrum, suggesting that the label was attached to at least two different binding sites. Using specific blocking reagents prior to spin labeling, the two binding sites were attributed to the sulfhydryl group of beta-93 (immobile) and the alpha-amino group of the N-terminal valines (mobile). The relative motion of the spin at one set of binding sites was restricted regardless of the state of ligation and pH, while the motion at the other site showed dependence on those parameters, e.g. the spin-labeled N-terminal ends of deoxyhemoglobin have restricted motion at all pH ranges studied, while those of oxyhemoglobin are relatively free to move at the basic pH range, but become more restricted in the acidic pH range.

The behavior of holo- and apo-forms of bovine superoxide dismutase at low pH. 1. Holo-superoxide dismutase from bovine erythrocytes has been shown to undergo a reversible structural modification in the pH 3-5 range. 2. The spectral alterations observed on changing from neutrality to pH 2 were: a slight attenuation of the 680 nm absorbance; the loss of the 450 nm shoulder, apparent in the optical spectrum of the native protein; and a new band appeared at 330 nm. The circular dichroism at 600 nm was essentially lost while a weak negative band appeared at approx. 380 nm and a positive band at 310 nm. 3. The EPR spectrum was also modified on changing from the native to the low pH form: A parallel increased from approximately 130 to approximately 150 G, g parallel remained unchanged at approximately 2.27, and gm decreased from approximately 2.09 to approximately 2.08. The apparent linewidth remained essentially constant. 4. High resolution (220 MHz) PMR spectra of holo- and apoproteins revealed that the metals influence the three-dimensional structure of the protein. 5. PMR studies indicated that at pH 3 the apoprotein existed almost entirely in a random coil form and that it assumed a compact well-ordered structure on returning to neutral pH. The holoprotein maintained a compact, apparently dimeric, structure even at pH 3.

Physical properties and subunits of Haemopis grandis erythrocruorin. The erythrocruorin of the leech Haemopis grandis possessed an S20,w of 57 S at neutral pH, its isoelectric point at pH 6.0 and exhibited a slightly sigmoid oxygenation curve with n approximately 2.1 and P50 = 11.2 mm at pH 7.4. A minimum molecular weight of 24000 +/- 1500 per heme group was determined from the iron and heme contents, 0.22 +/- 0.01 and 2.73 +/- 0.14 weight %, respectively. The subunit composition of the erythrocruorin was investigated using gel filtration in sodium dodecyl sulfate and polyacrylamide gel electrophoresis in sodium dodecyl sulfate at neutral pH. Haemopis erythrocruorin dissociated in the presence of sodium dodecyl sulfate into four subunits (1 through 4) possessing molecular weights of about 27000, 23000, 21000 and 13500, respectively. When the erythrocruorin was reduced with mercaptoethanol prior to sodium dodecyl sulfate electrophoresis, three subunits were observed, possessing molecular weights of about 13000 (I), 16500 (II) and 28000 (III). Sodium dodecyl sulfate electrophoresis of the isolated subunits 1 through 4 showed that subunit I was provided by subunits 1 and 4, subunit II was provided by subunit 1 and subunit III was provided by both subunit 2 and subunit 3. Haemopis erythrocruorin thus appeared to consist of at least five different polypeptide chains. It is likely that not all of the constituent polypeptide chains were associated each with a heme group. The shape of the Haemopis erythrocruorin observed by electron microscopy appeared to be consistent with the two-tiered hexagonal array characteristic of annelid erythrocruorins and chlorocruorins.

Myosin from arterial smooth muscle: isolation following actin depolymerization. The contractile proteins from arterial smooth muscle are highly soluble, and can be extracted at I = 0.05. However, they can be precipitated by a prolonged dialysis at pH 6 to give an actomyosin with a high, although variable, actin:myosin ratio. The sedimentation behavior of this actomyosin at high ionic strength was examined as a function of pH, protein concentration and composition by preparative ultracentrifugation. Comparisons with synthetic skeletal muscle actomyosins of similar composition demonstrated significant differences in the behaviors of these two systems. It was found that much smooth muscle actomyosin is not dissociated by normally relaxing conditions, and that it sediments at a slower rate than F-actin. The solubility of the supernatant protein (a myosin-enriched actomyosin) in 0.2 M K Cl (pH 7) depended on the pH during centrifugation. A lower solubility was associated only with a higher actin concentration in the supernatant, suggesting a dependence on actin repolymerization. Pure myosin was selectively precipitated from the supernatant by polyethylene glycol-6000, but only when the protein was soluble at low ionic strength. The solubility of purified myosin was similar to that of myosin from striated muscles. A relationship between the presence of depolymerized actin and the high solubility of smooth muscle contractile proteins is suggested.

Effects of membrane ribonuclease and 3'-nucleotidase on the digestion of polyuridylic acid by rat liver plasma membrane. 1. Fragments of isolated rat liver plasma membrane possess a ribonuclease activity which at pH 7.8 in the presence of 10 mM EDTA can digest polyuridylic acid (poly(U)) and polycytidylic acid (poly(C)) but not polyadenylic acid (poly(A)) and polyguanylic acid (poly(G)). Under these conditions, the membrane preparation does not degrade native or denatured DNA. 2. The products of the reaction with poly(U) (10 mM EDTA present) can be separated on DEAE-Sephadex into oligonucleotides of increasing chain length. Most of the products are di- to hexa-nucleotides which contain terminal 3'-phosphate groups. 3. When EDTA is not present (pH 7.8 or 8.8) the plasma membrane preparation degrades both poly(A) and poly(U). With poly(A) the product is all nucleoside while with poly(U) as substrate most of the product is nucleoside, but also some oligonucleotides are produced. 4. The ribonuclease releases acid soluble products very slowly from high concentrations of poly(U) (mg/ml). 5. Uridine trinucleotide with and without a terminal 3'-phosphate group is degraded by rat liver plasma membrane. The trinucleotide diphosphate is rapidly hydrolyzed to nucleoside while the trinucleotide itself is slowly digested and yields intermediate products, including nucleoside.

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+. The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.

Conformational and molecular responses to pH variation of the purified membrane adenosine triphosphatase of Micrococcus lysodeikticus. A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95% pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 degrees C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10(-4) M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20-30% of the activity could be recovered when the pH was returned to 7.5. In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel electrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.

Membrane-bound enzymes. III. Protease activity in leucocytes in relation to erythrocyte membranes. Protease activity was detected in membranes of human bovine erythrocytes prepared by the conventional procedures which include washing and removal of the \"buffy layer\". The enzyme was extracted by 0.75 M KCNS or (NH4)2SO4 and was activated by 0.4 to 0.5 M of the same salts. Colored, particulate hide powder-azure, membrane fractions and soluble proteins such as hemoglobin, casein or albumin were susceptible to hydrolysis by the membraneous protease. Partial purification of the enzyme was accomplished through disc-gel electrophoresis on polyacrylamide in the presence of 0.25% positively charged detergents like cetyltrimethylammonium bromide. An alkaline protease (pH 7.4) with properties similar to those of the erythrocyte enzyme was found in leucocytes. The similarity between the properties of the leucocytic and erythrocytic proteases and the correlation of the activity in erythrocyte membranes with content of white cells in these preparations, suggest that enzymatic activities in the contaminating leucocytes are responsible for the activity of membraneous proteases in erythrocytes.

[Relation between fluorescence and circular dichroism of the complex of the fluorescence probe 4-dimethylaminochalcone with serum albumin]. The fluorescence probe(4-dimethylaminochalcone; DMH) was noncovalently linked to human serum albumin (HSA). The variation of pH was due to serum albumin structural changes, which was determined in terms of DMH and HSA fluorescence and CD spectra. Considerable changes of fluorescence and CD spectra were observed at pH 8 and 10, where there is ionization of two more recently titrated tyrosin residues. It is assumed that these two tyrosine residues are in binding region and quench the fluorescence of DMH between pH 4 to 8. Quenching disappears if these residues are ionized (pH greater than 8) or if the protein undergoes the N -F transition (pH less than 4).

[Level of nicotinamide coenzymes in the myocardium of rats during the effects of methylxanthines (theophylline, theobromine, caffeine) and catecholamines]. It was shown in acute experiments on rats that one hour after an intraperitoneal injection of theophylline (50 mg/kg) there was a decrease in the NAD + NADP content by 19.4%, a tendency to a fall of NAD.H2 + NADP.H2 was expressed, and the total nicotinamide coferment level was reduced. A tendency to decrease NAD + NADP and the total pyridine nucleotide level was seen after caffeine administration. The action of catecholamines and methylxanthines was compared. Theobromine produced no significant effect on the indices under study. It was shown that isadrine decreased the NAD + NADP level; adrenaline (25 mkg/kg) increased the content of both the oxidized (by 24%) and of the reduced (by 48%) forms of pyridine nucleotides. An increase of adrenaline dose to 1000 mkg/kg was accompanied by reduction of the oxidized forms (by 22.2%) and of the total nicotinamide coferment level (by 18%).

Potable water quality in rural Georgetown County. Drinking water supplies of 161 rural communities, in Georgetown County, South Carolina, were randomly selected for sample collection. The analysis showed that most of the waters were slightly acidic. Low, but acceptable concentrations of chloride, copper, fluoride, sodium, cadmium, nitrate and phosphate were found. A few water samples showed higher then recommended levels of arsenic, mercury, zinc and lead. Although only 2% of the samples exceeded the mandatory limit of 0.05 ppm for arsenic, 72% exceeded the recommended level of 0.01 ppm. The mandatory limit for manganese was exceeded in 37% of the waters while 88% exceeded the limit for iron. The high iron content was generally responsible for the high turbidity found in 45% of the samples. The well depth and the consumer income had some bearing on water quality. Statistical evidence suggested that septic tank seepage was partially responsible for nitrate, phosphate, iron and arsenic contamination of shallow water supplies. Lead concentrations appear to vary according to the plumbing used and the pH of the waters.

The importance of an innervated and intact antrum and pylorus in preventing postoperative duodenogastric reflux and gastritis. This study has investigated the relationship between duodenogastric reflux, gastritis and certain symptoms 6-12 months after three operations for uncomplicated duodenal ulcer. The operations studied were proximal gastric vagotomy (PGV, 20 cases), truncal vagotomy and pyloroplasty (TV+P, 22 cases) and truncal vagotomy and antrectomy (TV+A, 21 cases). Duodenogastric reflux was assessed both by a radiological technique and by measuring the concentration of bilirubin in the gastric aspirate before and after operation. Incidence and severity of postoperative gastritis were determined by endoscopic biopsy. Symptoms were assessed by symptomatic score and Visick grading. There was a significant correlation between duodenal reflux and histological evidence of both severe superficial gastritis and glandular atrophy (P less than 0-01). There was also a close association between the degree of reflux and the presence of severe heartburn, epigastric pain and bile vomiting after operation. The amount of reflux did not differ before operation. There was significantly less reflux following PGV than after either TV+P (P less than 0-025) or TV+A (P less than 0-001). The results indicate that an operation which preserves an innervated and intact antrum and pylorus will protect against postoperative duodenogastric reflux, gastritis and symptoms.

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia. A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.

Endoscopic papillotomy and removal of gall stones. Endoscopic papillotomy was attempted in 59 patients with extrahepatic obstruction of the biliary duct system and was actually performed in 50 patients. A special high-frequency diathermy knife was introduced via a duodenoscope into the terminal common bile duct and the roof of the papilla was incised. In 33 out of 39 patients with choledocholithiasis the stones passed into the duodenum spontaneously or were removed endoscopically. Papillary stenosis without ductal stones was successfully treated with this method in eight out of 11 patients. One perforation of the duodenocholedochal junction occurred and was repaired surgically. Endoscopic papillotomy and stone extraction is a relatively safe and effective method of treating extrahepatic jaundice.

Inhibitory postsynaptic actions of taurine, GABA and other amino acids on motoneurons of the isolated frog spinal cord. The actions of glycine, GABA, alpha-alanine, beta-alanine and taurine were studied by intracellular recordings from lumbar motoneurons of the isolated spinal cord of the frog. All amino acids tested produced a reduction in the amplitude of postsynaptic potentials, a blockade of the antidromic action potential and an increase of membrane conductance. Furthermore, membrane polarizations occurred, which were always in the same direction as the IPSP. All these effects indicate a postsynaptic inhibitory action of these amino acids. When the relative strength of different amino acids was compared, taurine had the strongest inhibitory potency, followed by beta-alanine, alpha-alanine, GABA and glycine. Topically applied strychnine and picrotoxin induced different changes of post-synaptic potentials, indicating that distinct inhibitory systems might be influenced by these two convulsants. Interactions with amino acids showed that picrotoxin seletively diminished the postsymaptic actions of GABA, while strychnine reduced the effects of taurine, glycine, alpha- and beta-alanine. But differences in the susceptibility of these amino acid actions to strychnine could be detected: the action of taurine was more sensitively blocked by strychnine compared with glycine, alpha- and beta-alanine. With regard to these results the importance of taurine and GABA as transmitters of postsynaptic inhibition on motoneurons in the spinal cord of the frog is discussed.

The involvement of lysophosphoglycerides in neurotransmitter release; the composition and turnover of phospholipids of synaptic vesicles of guinea-pig cerebral cortex and Torpedo electric organ and the effect of stimulation. (1) Crude synaptosomal fractions (P2) derived from guinea-pig cerebral cortex were incubated in the presence of 50 mM KCl in a Krebs-glucose medium. Torpedo marmorata electric organs were stimulated electrically in vivo at 5 pulses/sec for 30 min by electrodes placed on the electric lobe. Synaptic vesicles were isolated from each source and the phospholipid compositions analysed and compared with vesicles from unstimulated controls. (2) Lysophosphatidylcholine was the only lysophosphoglyceride demonstrable in the synaptic vesicles from either source and its low levels did not increase as a result of chemical or electircal stimulation. In each case there was a close similarity of the phospholipid distributions in the vesicles taken from control and stimulated samples. (3) Control experiments indicated extensive decreases in the acetylcholine content of the vesicles from the stimulated electric organ and smaller decreases in the acetylcholine content of the synaptic vesicles from stimulated crude synaptosomal fractions. These fractions were found to respire linearly in the presence of 10 mM glucose and the vesicle fractions were shown to have low levels of contaiminating membranes as judged by marker enzyme analyses. (4) Crude synaptosomal fractions from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium with labelled fatty acids and [3H]glucose in the presence or absence of 50 mM KCl. Subsynaptosomal fractionation was carried out and specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomes) and H (disrupted synaptosomes). The release of neurotransmitter did not significantly enhance the labelling of phospholipids in any of the fractions studied as compared with phospholipids from unstimulated fractions. This was found after two incubation times and using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose.

The prevention of autolysis of stored cornea using steroid as a lysosome membrane stabilizer. Many eyes donated for use in corneal grafting are rejected because of signs of autolysis in the donor material. The purpose of this experimental study was to determine whether hydrocortisone acting as a lysosome membrane stabilizer could prevent or retard autolysis of the corneas under storage, and if so, what was the most efficacious concentration. Different groups of rabbit corneas were placed in saline as controls or in varying concentrations of hydrocortisone (10(-10) M to 10(-4) M at pH 7.4) at 37 degrees C and 4 degrees C. Acid phosphatase released after six hours was measured biochemically. This enzyme was used as a marker enzyme reflecting lysosomal labilization. Results showed a significant stabilization of the lysosomal membrane at 4 degrees C as compared to 37 degrees C. A trend towards stabilization of the lysosomal membrane was seen when 10(-8) M concentration of hydrocortisone at 37 degrees C was used, there being no demonstrable stabilization at 4 degrees C.

Respiratory effects of H+ and dinitrophenol injections into the brain stem subarachnoid space of fetal lambs. Mock cerebrospinal fluid (pH 5.37-8.38) or 2,4-dinitrophenol (DNP) (0.15-1.5 mg) was injected into the subarachnoid space of the ventral brain stem of exteriorized fetal sheep. Changes in pH on the ventral surface of the medulla did not stimulate respiratory efforts or induce significant cardiovascular changes. The respiratory response to DNP injections ranged from no response to prolonged rhythmic ventilation that was independent of the peripheral chemoreceptors or the control arterial pH and blood gas tensions. This inconsistency suggests an effector site somewhat removed from the immediate surface of the medulla. The heart rate and blood pressure were not affected. It is concluded that increased H+ concentration in the extracellular fluid of the fetal ventral medulla does not initiate respiration, and any respiratory response to metabolic inhibitors applied to this area therefore is not attributable to a secondary change in surface pH.

Soil fungistasis: elevation of the exogenous carbon and nitrogen requirements for spore germination by fungistatic volatiles in soils. Axenic, washed conidia of Fusarium solani f. sp. phaseoli, Aspergillus flavus, and Verticillium albo-atrum were placed on washed Difco purified agar discs along with an inorganic salt solution containing various levels of carbon and nitrogen substrates. These discs were exposed to volatiles from six soils (pH 5.1-8.6). Fusarium solani macroconidial germination was inhibited mostly by volatiles from soils of pH 5.1, 6.1, 7.0, and 7.5, but high levels of glucose and NH4Cl reversed this inhibition, raising germination to that of no-soil, no-carbon or nitrogen controls. Conidial germination of A. flavus was inhibited mainly by volatiles from high pH (7.0, 7.8, and 8.6) soils, and increased levels of glucose plus an amino acid mixture nullified this inhibition. Volatiles from soils of pH 5.1, 6.1, and 7.5 stimulated A. flavus conidial germination. Assays after the removal of CO2 from the air above soil of pH 5.1 demonstrated that volatiles inhibitory to A. flavus were produced by this soil. Assays indicated that a KOH-soluble compound was a fungistatic soil volatile to F. solani macroconidial germination. The nullification by carbon and nitrogen substrates of F. solani and A. flavus inhibition caused by soil volatiles parallels that for soil fungistasis. Conidial germination of V. albo-atrum was markedly stimulated by volatiles in all soils tested, and was not affected by removal of CO2. Inhibitory soil volatiles may increase the nutritional requirements for spore germination of certain fungi.

Nitrofurazone-reducing enzymes in E. coli and their role in drug activation in vivo. Earlier work showed that Escherichia coli contains at least two enzymes which reduce nitrofurazone and other nitrofuran derivatives. One of these enzymes is lacking in some nitrofurazone-resistant mutant strains. We now report that there are three separable nitrofuran reductases in this organism: reductase I (mol. wt. approximately 50 000, insensitive to O2), reductase IIa (mol. wt. approximately 120 000, inhibited by oxygen), reductase IIb (mol. wt. approximately 700 000, inhibited by O2). Unstable metabolites formed during the reduction of nitrofurazone by preparations containing reductases IIa and IIb produce breaks in DNA in vitro. In vivo experiments with nitrofurazone-resistant strains, which lack reductase II but contain reductases IIa and IIb, demonstrated that lethality, mutation, and DNA breakage are all greatly increased when cultures are incubated under anaerobic conditions, i.e., conditions such that reductase II is active. These results provide further evidence for the importance of reductive activation of nitrofurazone.

Physiological differences among isolates of Phytophthora cinnamomi. Significant differences in amylase, beta-glucosidase, and phosphatase activities were observed among four Phytophthora cinnamomi isolates grown in nutrient-amended sterilized soil for 20 days. Amylase pH optima for the four isolates were within a relatively narrow range; at pH 5.5 each isolate was within 90% of its peak activity. Isolates SB-216-1, 1-281, and C-39 each exhibited maximal beta-glucosidase activity at pH 5.0 and maximal phosphatase activity at pH 5.0-5.5. Maximal activity for these two enzymes of isolate A-7725 occurred at pH 3.5. In timed experiments, isolates 1-281 and A-7725 exhibited greater amylase activities than did the other two isolates. For beta-glucosidase, greatest activity was observed for SB-216-1; ACTivity of 1-281 was intermediate and least activity was observed for isolates A-7725 and C-39. Isolates SB-216-1 and 1-281 exhibited greatest phosphatase activities; isolate C-39 was intermediate in activity, and A-7725 was least active. Results indicate that significant differences exist among the isolates tested and that these differences can be quantitatively measured by the methods described.

Regulation and properties of an invertase from Clostridium pasteurianum. An intracellular invertase was induced in cultures of Clostridium pasteurianum utilizing sucrose as its carbon source for growth. This enzyme synthesis could be repressed by the addition of fructose of a sucrose-growing culture. In contrast, invertase activity was not affected by the addition of glucose to sucrose-growing cells and this enzyme could be induced in a glucose-metabolizing culture by the addition of sucrose. This enzyme was purified 10.5-fold over the induced lese, EC 3.2.1.26) by substrate-specificity studies. Invertase had a pH optimum of 6.5 and an apparent Km of 79.5 mM for sucrose, and required high concentration of potassium phosphate for maximum activity. Invertase was completely inactivated by a 2-min heat treatment at 60 degrees C. This enzyme was strongly inhibited by p-hydroxymercuribenzoate (pCMB) and weakly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), while cysteine could substantially reverse pCMB) inhibition, suggesting that sulfhydryl group(s) were necessary for invertase activity.

Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotobacter vinelandii. The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azotobacter vinelandii concentrates in the membranous R3 fraction that is directly associated with Azotobacter electron transport function. Sonically disrupted Azotobacter cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R3 membranous fraction which sediments on ultracentrifugation at 144 000 X g for 2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activty occurred at pH 8.0; Mg2+ ion when added to the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATP stoichiometry was 1:1 on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0-60 min). The membrane-bound ATPase in the R3 fraction was stimulated 12-fold by treatment wiTH TRypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R3 electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R3 fraction had no effect on activity; overnight refrigeration at 4 degrees C, however, resulted in a 25% loss of activity as compared with a 14% loss when the R3 fraction was stored overnight at 25 degrees C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (-20 degrees C), and subsequent sonication failed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R3 electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation.

Effects of zinc-smelter emissions on forest soil microflora. Within 2 km of a zinc (Zn) smelter in Palmerton, Pennsylvania, near the Lehigh Water Gap, up to 13.5% Zn by weight has been measured in the O2 horizon of the soil, and up to 8% Zn in the A1 horizon. The total numbers of bacteria, actinomycetes, and fungi (measured by dilution plate counts) were greatly reduced in the most severely Zn-contaminated soils compared with control soils. The reduction of microbial populations may be a partial cause of the decreased rate of litter decomposition at Lehigh Gap. Growth of most bacteria from control sites was reduced by 100 to 200 muM Zn, most actinomycetes by 100 muM Zn, and most fungi by 100 to 1000 muM Zn in thin-Pablum extract agar (TPab). All the tested actinomycetes and non-spore-forming bacteria isolated from Zn-contaminated Lehigh Gap soils were Zn-tolerant, growing normally in media containing 600-2000 muM Zn. Most fungi, regardless of source, were capable of at least 50% of normal growth at 700 muM Zn. Zinc-tolerant bacteria, actinomycetes, and fungi were readily isolated from low-Zn soils, suggesting that selection for Zn tolerance may proceed rapidly. Acidophilic Mortierella species have been selectively eliminated near the smelter, apparently because of elevated soil pH. Peryronellaea glomerata (Corda) Goidanich and Coniothyrium spp. were found only in the high-Zn soils.

The effects of the continuous administration of N,N-dimethyl-4-phenylazoaniline (DAB) on the activities and the inducibilities of some drug-metabolizing enzymes in rat liver. (1) The effect of feeding a relatively low-protein diet containing 0.06% DAB for 29 weeks on the activity of DAB-azoreductase, nitroreductase (p-nitrobenzoic acid), N-oxidase (N,N-dimethylaniline), N-demethylase (DAB), cytochrome P-450, NADPH-cytochrome c reductase, beta-glucuronidase and arylsulphatase A were studied. Rapid decreases occurred in the activities of the first six enzymes, reaching minimal values at between 4 and 8 weeks. Activities then increased in all cases to control or nearly control levels. This rate of increase was least for cytochrome P-450. At 4 weeks azoreductase activity with the chemotherapeutic agent CB10-252 (I) as substrate was significantly higher than in control rats. Early increases occurred in the activities of beta-glucuronidase and arylsulphatase A and the activity of the latter never dropped below the control level. (2) An investigation was made of the differential effects of dye feeding on some of the enzyme activities in the two major liver lobes and differences were found. (3) The effect of phenobarbital (PB) pretreatment on the DAB-fed rats was studied at 4-week intervals. The activities of DAB-azoreductase and of nitroreductase increased throughout the whole period, while the activities of the lysosomal enzymes were decreased. (4) After feeding DAB for 4 weeks the effect of PB and 3-methylcholanthrene (MC) on the activities of DAB-azoreductase, CB10-252-azoreductase and components of the azoreductases-cytochrome P-450, NADPH-cytochrome c reductase, the CO-CB10-252-azoreductase was not induced by PB or MC, and CO did not inhibit its reduction. Its reduction depended only slightly on NADH. CO caused a greater relative decrease in the activity of DAB-azoreductase in dye-fed animals and also in animals following PB and MC pretreatment, implying a greater role of cytochrome P-450 in dye-fed animals.

Some characteristics of two azoreductase systems in rat liver. Relevance to the activity of 2-[4'-di(2\"-bromopropyl)-aminophenylazo]benzoic acid (CB10-252), a compound possessing latent cytotoxic activity. The system involved in the reduction of 2-[4'-di(2''-bromopropyl) aminophenylazolbenzoic acid (CB10-252), an agent designed for treating primary liver cell cancer, has been demonstrated to be localised mainly in the 108 000 X g supernatant fraction of rat liver homogenate. It is also present in other organs particularly in the spleen. DAB-azoreductase as shown previously is present almost entirely in the microsomal fraction and is found in high concentration only in liver. The pH maximum for CB10-252-azoreductase implying the importance of the 2'-carboxyl group in determining substrate specificity. The use of enzyme inhibitors and other additives showed that CB10-252 WAS NOT AXANTHINE OXIDASE OR DIHYDROFOLATE REDUCTASE. Its activity was not affected by carbon monoxide, phenobarbitone (PB), or 3-methylcholanthrene (MC) pretreatment. Enhancement of the activity by ferrous ions and FAD indicated that at least part of the reduction system could involve a flavoprotein with FAD as the prosthetic group. The activity of CB10-252-azoreductase and methylred-azoreductase was reduced by menadione (vitamin K3), cyanide and propylgallate. A diaphorase preparation from pig heart reduced both CB10-252 and methylred with both NADPH- and NADH-generating systems.

Mercury inhibition of avian fatty acid synthetase complex. (1) Subcutaneous or intra-abdominal injections of 8 mg of HgCl2/100 g body weight markedly depressed hepatic fatty acid synthetase activity of chicks at 1 h post-injection. The depression occurred despite the fact that the chicks continued to eat up until the time they were killed. Under these same conditions, the hepatic activity of acetyl-CoA carboxylase (EC 6.4.1.2) was not affected by HgCl2, while the activity of the mitochondrial system of fatty acid elongation was stimulated. (2) When 2-mercaptoethanol was included in the incubation medium for a highly purified preparation of fatty acid synthetase, 500 muM HgCl2 was required to show definite inhibition of the enzyme. When 2-mercaptoethanol was omitted, 50 muM HgCl2 was inhibitory and 100 muM HgCl2 abolished enzyme activity. (3) 2 mM dithiothreitol completely protected the purified fatty acid synthetase preparation from inhibition by 100 muM HgCl2. When dithiothreitol was added after the addition of enzyme to the mercury-containing medium, protection of the enzyme was not complete. (4) Dialysis of cytosol fractions from chicks injected with HgCl2 against 500 vol. of 0.2 M potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 10 mM dithiothreitol for 4 h at 4 degrees stimulated the fatty acid synthetase activity of the fractions. Dialysis of cytosol fractions from noninjected chicks under the same conditions was without effect on fatty acid synthetase activity. (5) These data support the hypothesis that the inhibitory effect of HgCl2 administered in vivo on hepatic fatty acid synthetase activity in chicks is mediated through the interaction of mercury with the sulfhydryl groups of the enzyme.

Effects of decreasing arterial blood pressure on cerebral blood flow in the baboon. Influence of the sympathetic nervous system. The influence of the sympathetic nervous system on the cerebral circulatory response to graded reductions in mean arterial blood pressure was studied in anesthetized baboons. Cerebral blood flow was measured by the 133Xe clearance method, and arterial blood pressure was decreased by controlled hemorrhage. In normal baboons, the constancy of cerebral blood flow was maintained until mean arterial blood pressure was approximately 65% of the base-line value; thereafter, cerebral blood flow decreased when arterial blood pressure was reduced. Superior cervical sympathectomy of 2-3 weeks duration did not affect the normal response. In contrast, both acute surgical sympathectomy (cervical trunk division) and alpha-receptor blockade (1.5 mg/kg of phenoxybenzamine) enhanced the maintenance of cerebral blood flow in the face of hemorrhagic hypotension in that cerebral blood flow did not decrease until mean arterial blood pressure was approximately 35% of the base-line value. The results indicate that the sympathetic nervous system is not involved in the maintenance of cerebral blood flow in the face of a fall in arterial blood pressure. Indeed, the implication is that the sympathicoadrenal discharge accompanying hemorrhagic hypotension is detrimental to, rather than responsible for, cerebral autoregulation.

Influence of hematocrit, blood gas tensions, and pH on pressure-flow relations in the isolated canine lung. An isolated perfused canine lung preparation in which determinants of vascular caliber could be individually controlled was developed. The relation of pulmonary arterial (Pa), venous (PV), and alveolar (PA) pressures was such that Pa greater than PA greater than PV throughout the whole lung. The addition of isoprenaline to the perfusate abolished vascular reactivity. Once stability was reached, vascular cross-sectional area remained acceptably constant for 2.25 hours as judged by normalized conductance. The influence of perfusate hematocrit, blood gas tensions, and pH on pressure-flow relations was then studied in 15 isolated canine lungs. The hematocrit-vascular conductance relation was derived at constant perfusion pressure. Conductance varied linearly with hematocrit over a range of 16.5 to 89.5%. Mean pulmonary arterial blood gas tensions were: PO2 = 121 mm Hg, PCO2 = 28 mm Hg, and pH = 7.46. Acute respiratory acidosis (PO2 = 30 mm Hg, PCO2 = 81 mm Hg, pH = 7.17) and lactic acidosis and hypoxemia (PO2 = 32 mm Hg, PCO2 = 21 mm Hg, pH = 6.96) did not significantly alter this relation. Transformation of the conductance-hematocrit data indicated that hematocrit was the most important determinant of relative apparent viscosity of the blood. Both acute respiratory and lactic acidosis failed to significantly increase relative viscosity within the range of hematocrit usually found in secondary polycythemia.

Interaction of the chemoreflex and the pulmonary inflation reflex in the regulation of coronary circulation in conscious dogs. The interaction of chemoreflex and pulmonary inflation reflex control of the coronary circulation was examined in conscious dogs by comparing the responses to chemoreflex stimulation (intracarotid injection of nicotine) when ventilation was allowed to increase with those when ventilation was controlled. The responses were also compared with those elicited by both forced mechanical and spontaneous hyperinflation. When the heart rate was constant, intracarotidly administered nicotine induced an increase in the depth of respiration followed closely by an increase in late diastolic coronary flow from 48 +/- 2 to 106 +/- 8 ml/min and a reduction in late diastolic coronary resistance from 1.62 +/- 0.08 to 0.78 +/- 0.06 mm Hg/ml min-1. After beta-receptor and cholinergic blockade, a similar coronary dilation in response to nicotine occurred only when ventilation was allowed to increase. However, when ventilation was controlled, intracarotidly administered nicotine increased coronary resistance after combined beta-receptor and cholinergic blockade. The reflex coronary dilation was not observed after carotid sinus nerve section or after alpha-receptor blockade. Thus, nicotine stimulation of the carotid chemoreflex results in a striking coronary dilation that has two components. The minor component involves a chemoreflex with its efferent pathway in tthe vagi. The major component of coronary dilation follows an increase in the depth of respiration, and its efferent component appears to involve withdrawal of alpha-adrenergic constrictor tone. An almost identical period of reflex coronary dilation followed either forced mechanical or spontaneous hyperinflation in the conscious dog.

Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts. The rate of coronary blood flow was varied in isolated working rat heart preparations to determine its influence on the rate of glocose utilization, tissue high-energy phosphates, and intracellular pH. A 60% reduction in coronary blood flow resulted in a 30% reduction in oxygen consumption, an accelerated rate of glusoe utilization, lower tissue levels of high-energy phosphate, and higher tissue levels of lactate and H+. Ventricular performance deteriorated as reflected by a decrease in heart rate and peak systolic pressure. Further reductions in coronary blood flow resulted in inhibition of glycolysis, a greater decrease in tissue levels of high-energy phosphates, and higher tissue levels of both lactate and H+. These changes in glycolytic flux, tissue metabolites, and ventricular performance were proportional to the degree of restriction in coronary blood flow. The importance of coronary blood flow and washout of the interstitial space in the maintenance of accelerated glycolytic flux in oxygen-deficient hearts is emphasized. It is concluded that acceleration of ATP production from glycolysis can occur only in the marginally ischemic tissue in the peripheral area of tissue supplied by an occluded artery. The central area of tissue which receives a low rate of coronary blood flow will have a reduced rate of ATP production due to both a lack of oxygen and an inhibition of glycolysis.

Mechanisms of glycolytic inhibition in ischemic rat hearts. The mechanisms of glycolytic inhibition in ischemic myocardium were investigated in the isolated, perfused rat heart. Glycolysis was inhibited at the level of glyceraldehyde-3-phosphate dehydrogenase. The major factors that accounted for the glycolytic inhibition in the ischemic heart compared with the anoxic heart appeared to be higher tissue levels of lactate and H+ in the ischemic tissue. Increased extracellular pH inhibited glycolysis in anoxic and hypoxic hearts much more readily than it did in aerobic hearts. However, maintenance of both extracellular and intracellular pH caused only a modest acceleration of glycolysis in ischemic hearts. Accumulation of tissue lactate and inhibition of glycolysis were directly proportional to the reduction in coronary bloow flow in both anoxic and ischemic hearts. At intracellular lactate concentrations between 15 and 20 mM, glycolysis was inhibited under both conditions. Addition of either 10, 20, or 40 mM lactate to the perfusate inhibited glycolysis in aerobic, anoxic, and ischemic hearts. The effect of lactate did not appear to be mediated through changes in intracellular pH. It is concluded that accumulation of lactate represents a major factor in the inhibition of glycolysis that develops in ischemic hearts.

Comparison of contractile performance of canine atrial and ventricular muscles. This study compared the contractile performance of a canine right atrial trabecula with that of a macroscopically indistinguishable trabecula isolated from the right ventricular apex. The heart was removed from nine mongrel puppies weighing 6-8 kg and placed in Krebs-Ringer's bicarbonate solution. The bathing solution contained only 1.25 mmoles of Ca2+ and was bubbled with a 95% O2-5% CO2 gas mixture. Each atrial trabecula was specially selected from the right atrial appendage. Histologically, these trabeculae showed a remarkable longitudinal orientation of the fibers. At Lmax (the length of the muscle at which developed tension was maximum) under identical conditions of temperature, rate of stimulation, ionic milieu, pH, and O2 and CO2 supply, right atrial trabeculae achieved the same developed and total tensions but in a much shorter time than did ventricular trabeculae. In both muscle groups the maximum developed tension averaged about 2.5 g/mm2. Since Lo (expressed as a fraction of Lmax) was less in atrial muscle than it was in ventribular muscle, we concluded that atrial muscle can be stretched considerably more than can ventricular muscle before optimum length is reached. At any given initial muscle length, the maximum of tension rise for atrial trabeculae amounted to at least twice that for ventricular trabeculae. At any given load up to 1.5 g/mm2, the maximum velocity of shortening of an atrial trabecula was about three to four times that of a ventricular trabecula. These results collectively indicate that the contractile performance of the right atrial muscle is in many respects superior to that of the right ventricle, at least under the conditions of these experiments.

Serum lactate dehydrogenase activity ratios with different substrates. 1. The lactate dehydrogenase activity of 89 sera from patients suffering myocardial infarction and of 55 sera from patients with hepatocellular damage was assayed under optimal conditions using pyruvate, alpha-oxobutyrate, hydroxypyruvate and glyoxylate as substrates. Activity was also measured with lactate as substrate at different pH values. 2. The ratios of activities under these different assay conditions were calculated for both series of patients. Correct differentiations for single ratios ranged from virtually nil for hydroxypyruvate/alpha-oxobutyrate to is greater than 93 per cent for glyoxylate/hydroxypyruvate and glyoxylate/alpha-oxobutyrate. This was little improved by the use of multiple ratios involving up to seven separate assays. 3. The activity ratio of hydroxypyruvate to pyruvate which is consistently greater than unity was found to be inverted in a case of morphine poisoning.

Rapid kinetic measurement of lactate in plasma with a centrifugal analyzer. In this method, blood is collected in ammonium heparinized microhematocrit tubes and lactate is directly determined in the plasma, separated within 15 min from the erythrocytes. Lactate is assayed by mixing 10 mul of sample with NAD+ and lactate dehydrogenase in tris(hydroxymethyl)aminomethane hydrazine buffer. The rate of increase in absorbance of the NADH formed, measured at 340 nm, is proportional to lactate concentration. The assay is complete in 4 min and absorbance is linearly related to concentration from 0.625 to 15 mmol/liter. Analytical recoveries of lactate added to plasma averaged 104% (range, 91-116%). Results compared well for plasma samples analyzed by this method with the CentrifiChem and the Du Pont aca.

Anaerobic glycolysis in normal human erythrocytes incubated in vitro with sodium salicylate. 1. Some effects of sodium salicylate upon anaerobic glycolysis have been studied in normal human erythrocytes incubated for up to 6 h at 37 degrees C in autologous sera. 2. Both glucose consumption and lactate production were stimulated by concentrations of salicylate up to 60 mmol/l but at the highest concentration used (90 mmol/l) an initial stimulus was followed by inhibition of glycolysis. 3. Losses occurred of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-phosphate(AMP)at higher concentrations of salicylate and there was a concomitant increase of inorganic phosphate. 4. Other phosphate esters underwent concentration changes at higher concentrations of salicylate that reflected inadequate concentrations of ATP for glycolysis. 5. The rates of sodium efflux from, and potassium influx into, erythrocytes were unaffected by the presence of salicylate at concentrations sufficient to stimulate glycolysis.

Arterial catecholamines in hypoxic exercise in man. 1. We measured the minute ventilation and arterial blood catecholamine concentrations in four normal men standing and at two levels of moderate treadmill exercise breathing 14% oxygen or air. 2. Minute ventilation was significantly higher during hypoxic exercise than during normoxic exercise at an oxygen uptake of 1500 ml/min. 3. Arterial plasma noradrenaline during hypoxic exercise at an oxygen uptake of 1500 ml/min was significantly greater than at rest. 4. Arterial plasma noradrenaline during normoxic exercise at an oxygen uptake of 1500 ml/min was not elevated above the resting concentration. 5. The results are compatible with the suggestion that increased concentrations of arterial plasma noradrenaline contribute to the hypoxic potentiation of the respiratory response to moderate exercise.

Effects of graded infusions of monomethylmethacrylate on coagulation, blood lipids, respiration and circulation. An experimental study in dogs. In 4 dogs injected intravenously (i.v.) with 125I labeled fibrinogen, 51Cr labeled platelets and 99mTc labeled albumin, and subjected to successively increasing amounts of i.v. infused monomethylmethacrylate, doses corresponding to the amounts released into the blood stream following implantation of acrylic cement during total hip replacements did not affect the clotting mechanism, did not cause trapping of platelets and fibrin in the lungs, did not generate fat emboli, and did not cause depression of the arterial oxygen tension or blood pressure. Monomethylmethacrylate in whole blood was associated with both blood cells and plasma.

Influence of pH and hypoxia on the success of defibrillation. Clinical impressions about the problem of defibrillation during states of acid-base imbalance and hypoxia have been influenced by studies involving the effect of these derangements on the ventricular fibrillation threshold. Based on body weight, energy requirements for defibrillation in normal dogs were compared to requirements in dogs subjected to commonly encountered acid-base disturbances and severe hypoxemia. No significant differences were found. Seventy-five percent of all animals in the study were electrically converted with low-to-moderate levels of energy. The incidence of spontaneous resumption of circulation following defibrillation was lowest in animals subjected to metabolic acidosis and hypoxia. The results suggest that pH and blood gas alterations, previously shown to influence the normal ventricular fibrillation threshold, do not significantly affect the normal defibrillation threshold.

Charge properties of human pituitary and amniotic fluid prolactins. The apparent isoelectric points (pI) in isoelectric focusing (IF) of human pituitary and amniotic fluid prolactin (hPRL), both non-iodinated and iodinated, were determined. Unresolved mixtures of pituitary hPRL isohormones E and F, and of at least five isohormones found in amniotic fluid, and plasma hPRL exhibit an average pI value of 6.5 - 6.7. Transient state pH values observed or previously reported for hPRL components range from pH 5.9 to 6.8 after correction to standard conditions. At pH 8.1, the major isohormone, hPRL-F, carriers a charge of 2.2 net protons per molecule. The net charge differences among isohormones E, F and G are compatible with acquisition or loss of single charged groups per 20,000 molecular weight. This net charge is similar to that of the least prolactin-bioactive major isohormone of human growth hormone (hGH-B), while the hGH with a bioactivity comparable to that of hPRL exhibits a net charge of 3.4 valence units. The \"large\" isohormones J and H increased net charges, by a factor of 2-3, in direct proportion to their size increments.

Human and monkey prolactin and growth hormone: separation of polymorphic forms by isoelectric focusing. The feasibility of using isoelectric focusing for the separation of primate pituitary growth hormone from prolactin and for the characterization of polymorphic forms of these hormones was explored. In a pH 3--10 gradient, extracts of both human and cynomolgus monkey pituitaries were each resolved into 4 growth hormone components and at least 3 prolactin components, as shown by radioimmunoassay. In narrower gradients (of 2--3 pH units) greater resolution was achieved; the principal growth hormone components were well separated from the principal prolactin components but there was overlapping of some minor components. A partially purified human prolactin preparation was found to contain 4 prolactin components, one of which had a prolactin/growth hormone ratio of 760. Clinical grade human growth hormone was also resolved into at least 5 prolactin and 5 growth hormone components, many of which had higher pI values than those found in pituitary extract. Under the conditions used, both growth hormone and prolactin were found to be polymorphic with respect to isoelectric point. Some of the human prolactin components were found to contain less than 0.2% growth hormone by radioimmunoassay. Monkey growth hormone containing 0.01% prolactin was isolated. These findings demonstrate that isoelectric focusing is useful for the preparation of both growth hormone and prolactin which are essentially free of one another. Furthermore, the polymorphic forms were repeatedly found in preparations obtained by several methods and from 2 different species, suggesting that these forms are not artifacts.

Ecological observation of the 137Cs-contamination in beef of animals from the southern-Bavarian area. Certain climatic and edaphic conformations in the Bavarian sub-alpine mountains and in the Alps favor above all the development of a land utilization system and farm structures similar to those in the northern part of Scandinavia. In 1963/64, the years of the highest environmental contamination up to the present, we established in 600 beef samples from the round or shoulder of male and female cattle (mainly Highland cattle) close connections between the 137Cs-contamination of green crop and the long lastnig yearly precipitation quantities, as well as certain relations between the 137Cs-contamination of meat and differences in the feeding and keeping of the animals. During summer-seasons (April-October), beef of cattle from pastures with heavy rainfall (Alps) was contaiminated by 137Cs up to 15 times more than that of confined animals. Hereby the rate of 137Cs-contamination in the meat of grazing cattle was nearly proportional to the quantities of precipitation. When confined cattle were fed on pastures in autumn after harvesting for 2 to 3 weeks, a quick increase of 137Cs-contamination of the meat was caused within this time up to values which in this district were otherwise only observed in grazing cattle. The lower 137Cs-content in meat of cattle housed during the summer season is due to the more varied fodder, which is at that time less contaminated than green crop. During the winter season (November to March), the highest contaminations in the meat of confined (Bohemian Forest) or grazing cattle (Alps) was measured when the animals in these districts were almost exclusively fed with fodder from the own farmground or with leafy silage. The highest contamination was almost regularly noticed in January, February and March, as generally during these months the highly contaminated first cut hay is fed. Here the meat was often even more contaminated than that of grazing cattle. After the quick decrease of 137Cs in fallout noticed in the years 1964 and 1965, in 1965/66 a dependance in the 137Cs-contamination of beef on the methods of keeping and feeding could still be observed in only the extreme cases (Alps, Bavarian- and Bohemian Forest); though in general, meat of animals from districts with heavy rainfall was slightly more contaminated than meat of animals from regions with less precipitation.

Liver microsomal beta-glucuronidase and UDP-glucuronyltransferase. Both UDP-glucuronyltransferase (GT) and beta-glucuronidase (betaG) were assayed in untreated liver microsomes. Optimum assay conditions were established with rat liver microsomes using p-nitrophenol (pNP) and its glucuronide (pNPGA) at the pH optima of GT (7.5) and betaG (4.5). The activities of the two enzymes were compared using microsomes from rats, mice, pigs, cattle and horses, with pNP, pNPGA, and phenolphthalein as substrate, in the presence of various cofactors and inhibitors at pH 7.5 and 4.5. These data disclose pronounced differences with respect to species, substrate and other experimental conditions, thereby precluding the establishment of general optimum conditions. The two enzymes were also assayed under strictly identical conditions using pNP and pNPGA and rat liver microsomes at pH 7.5 in the presence and absence of UDP-glucuronate disodium (UDPGA), activators (ATP;UDP-N-acetylglucosamine) and inhibitors. When provided with a functional level of UDPGA, both enzymes proved active under those conditions, and a conjugation-deconjugation interplay was indicated. The two processes could be selectively and totally inhibited by Zn2+ and saccharolactone. The results suggest that conjugation-deconjugation-reconjugation cycles may be operative in the metabolism of drugs in vivo, taking place already at the level of the liver endoplasmic reticulum.

Biochemical aspects of renal ammonia formation in metabolic acidosis. In omnivorous creatures, the diet is acidogenic, especially as a result of the meat content, which gives rise to phosphoric and sulphuric acids, i.e., to metabolic acidosis. In the short term, metabolic acids are buffered by tissue proteins and bicarbonate (the 'alkali reserve'). In the longer term, acid must be excreted, or neutralized with base which is also generated from the diet, by conversion of dietary amino-nitrogen to ammonia. The final steps of this process occur in the kidney, which converts circulating glutamine to ammonia, and to carbon products such as glucose and carbon dioxide, by metabolic reactions which adapt during acidosis to generate more ammonia and maintain an increased renal ammonia content. The complex mechanisms which govern the formation of ammonia, glucose and carbon dioxide from glutamine, involving the reactions of amino acids, the tricarboxylic acid cycle, and gluconeogenesis, are reviewed.

Studies on the mechanism of the changes in serum and liver gamma-glutamyl transpeptidase activity. I. Experimental extrahepatic cholestasis in rabbits. Serum, liver and renal gamma-glutamyl transpeptidase (GGT) activities were studied in four groups of rabbits: controls, rabbits with obstructive extrahepatic cholestasis, rabbits with obstructive anuria, and animals with combined obstructive extrahepatic cholestasis and obstructive anuria. Serum GGT was essentially increased in rabbits with obstructive extrahepatic cholestasis, showing peak values in the combined cholestasis + obstructive anuria group, and practically normal values in animals with anuria. Liver GGT was increased in both cholestasis groups, but the increase was less prominent than the increase in serum GGT and there was no correlation between them. In both anuric groups renal GGT was reduced, probably as a result of inhibited enzyme synthesis secondary to the altered conditions for adequate renal function. The results obtained are suggestive of a probable renal involvement in the formation of the serum GGT activity level.

Changes in erythrocyte 2,3 diphosphoglycerate in women following short term maximal exercise. 15 untrained women were subjected to a walking treadmill test to determine the influence of maximal exercise upon synthesis of erythrocyte 2,3 DPG. Although there was a 9.8% increase in the 2,3 DPG content following exercise, there was a concomitant 9.4% increase in the hemoglobin level; therefore, when 2,3 DPG is expressed as a ratio to hemoglobin (See Article), there was no significant change as a result of exercise stress. It was suggested that three additive factors produced during strenuous exercise; decreased pH; increased hemoglobin concentration; and increased CO2 production result in by-product inhibition of 2,3 DPG synthesis. It is concluded that 2,3 DPG does not provide a physiologic benefit in the adaptation of the oxygen transport system to exercise.

Hydroxylation of p-Coumaric acid by illuminated chloroplasts. The role of superoxide. 1. Chloroplasts isolated from leaves of spinach-beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p-coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction. 2. Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect. 3. Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2-mercaptoethanol. 4. It is proposed that O-2- generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.

Identification of chloride-binding sites in hemoglobin by nuclear-magnetic-resonance quadrupole-relaxation studies of hemoglobin digests. 35Cl minus-nuclear magnetic resonance (NMR) studies indicate that various digests of human hemoglobin with carboxypeptidase A and B, or a combination of the two, may be used for the identification of chloride binding sites. All the digestion products contain, like hemoglobin itself, at least two classes of binding sites, one of high, the others of low affinity. The pH dependence of the excess linewidth of the 35Cl minus NMR signal indicates that in the simple digests with either carboxypeptidase A or B, chloride is bound with high affinity at or near His-beta146-Asp-beta94 and at or near Val-alpha1-Arg-alpha141. The high-affinity sites show, in the case of the simple digests, a strong oxygen linkage which is lost in the forms digested with both carboxypeptidase A and B; this linkage may thus be correlated to the presence of conformational changes. Organic phosphates, like inositol hexaphosphate, show competition for some of the high-affinity chloride binding sites in hemoglobin and in the simple digests. This competition is likewise lost in the doubly digested hemoglobins.

The internal-alkaline pH gradient, sensitive to uncoupler and ATPase inhibitor, in growing Clostridium pasteurianum. 1. The intracellular pH was measured in growing Clostridium pasteurianum with and acid-base equilibrium distribution method. [14C]Dimethyloxazolidinedione, [14]methylamine and [14C]acetic acid were used as \"deltapH-indicators\". During growth the extracellular pH decreased from 7.1 to 5.1; simultaneously the intracellular pH changed from 7.5 to 5.9. Thus, the intracellular pH was more alkaline than the extracellular pH by 0.4 to 0.8 pH-units. 2. This pH gradient (interior alkaline) was abolished by the proton conductor carbonylcyanide m-chlorophenylhydrazone and the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The pH gradient could not be demonstrated in cells depleted of an energy substrate. These results suggest that the pH gradient is formed by an ATPase-driven extrusion of protons from the cells rather than by a Donnan potential. 3. Growth of the organism was inhibited by low concentrations of both carbonylcyanide m-chlorophenylhydrazone (5 muM) and dicyclohexylcarbodiimide (5 muM). This finding suggests that the pH gradient is essential for the growing cell as it may be required for substrate accumulation and other types of transport processes.

D-alanyl-D-alanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis. Membranes of the bacterial form and the stable and unstable L-forms of Proteus mirabilis contain LD and DD-carboxypeptidase. The DD-carboxypeptidase is inhibited non-competitively by penicillin G. The enzyme of the bacterial form is highly penicillin-sensitive (Ki - 4 X 10(-9) M penicillin G). Inhibition is only partly reversible by treatment with penicillinase or by dialysis against buffer. In contrast, the DD-carboxypeptidase of the unstable L-form, grown in the presence of penicillin, is 175-fold less penicillin-sensitive (Ki = 7 X 10(7) M penicillin G). Inhibition is completely reversed by penicillinase or dialysis. After inhibition by penicillin and subsequent reactivation the penicillin sensitivity of the bacterial DD-carboxtpeptidase is similar to the sensitivity of the enzyme of the unstable L-form. The hypothesis is proposed that P. mirabilis contains two DD-carboxypeptidases of different penicillin sensitivity and with different mechanisms of penicillin binding. Peptidoglycan synthesis in the cell walls of the unstable L-form is probably carried out with the help of only one DD-carboxypeptidase, viz. the completely reactivatable enzyme with the lower penicillin sensitivity.

Pigeon-liver NAD kinase. The structural and kinetic basis of regulation of NADPH. NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis-Menten behaviour with the substrate NAD+; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo.