bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular localization of γ-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain. Evidence for at least two small glutamate compartments in brain
Authors: Reijnierse G L , Veldstra H , Van den Berg C J
Published: August 2015

The subcellular localizations of γ-aminobutyrate transaminase (EC 2.6.1.19) and glutamate dehydrogenase (EC 1.4.1.2) in brain tissue of adult rats were compared with each other and with those of NAD+-isocitrate dehydrogenase (EC 1.1.41) and monoamine oxidase (EC 1.4.3.4; kynuramine as substrate). Crude mitochondrial fractions from brain tissue were centrifuged in continuous sucrose density gradients. γ-Aminobutyrate transaminase and glutamate dehydrogenase were always found at a higher density than NAD+-isocitrate dehydrogenase and monoamine oxidase. When centrifuged for 1 h at 53 000gav., there was a slight difference between the distribution profiles of glutamate dehydrogenase and γ-aminobutyrate transaminase. This difference was larger when the centrifugation time was only 15 min. It is concluded that there are subpopulations of brain mitochondria with differing proportions of γ-aminobutyrate transaminase and glutamate dehydrogenase. The results are discussed in relation to evidence obtained with labelled precursors in vivo that there are at least two small glutamate compartments in adult brain.
Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens
Authors: Hansma H G , Kung C
Published: August 2015

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.
The metabolism of lipids in mouse pancreatic islets. The biosynthesis of triacylglycerols and phospholipids
Authors: Berne C
Published: August 2015

The rate of incorporation of [U-14C]glucose and [u-14C]palmitate into the lipids of the pancreatic islets of obese-hyperglycaemic mice was examined. The following main observations were made. 1. Both glucose and palmitate were incorporated into lipids in the islets. The fraction of glucose utilized for lipid biosynthesis was calculated to be 3-6% of that oxidized at high and low glucose concentrations, whereas palmitate was about equally divided between oxidation and esterification into lipids. 2. Glucose was primarily incorporated from sn-glycerol 3-phosphate. Of the total glucose carbon incorporated, only 2-4% was recovered as fatty acids. 3. A major portion of both glucose and palmitate was incorporated into phospholipids, whereas 10-30% went into triacylglycerols, depending on the extracellular glucose concentrations. 4. An increase in the glucose concentration from 3.5 to 17 mM caused a twofold increase in the rate of glucose incorporation into triacylglycerols and a fivefold increase in the rate of incorporation into phospholipids. Similar effects were also obtained with normal mouse islets. Palmitate was also preferentially directed into phospholipids by an increased glucose concentration. 5. Islets pre-labelled with radioactive palmitate showed a decrease in triacylglycerol radioactivity when they were subsequently incubated in the absence of exogenous sources of energy. 6. Mannoheptulose inhibited the rate of glucose incorporation into phospholipids, whereas omission of Ca2+ and adrenaline left phospholipid biosynthesis unimpaired. The results suggest that pancreatic B-cells have the capacity to store and utilize energy in the form of triacylglycerols. A stimulation of the B-cells by glucose is followed by an increased rate of phospholipid biosynthesis. However, this does not seem to be directly linked to the release of secretory granules.
Purification and properties of pyruvate kinase from the hepatopancreas of Carcinus maenas
Authors: Giles I G , Poat P C , Munday K A
Published: August 2015

Pyruvatekinase from the hepatopancreas of the common shore crab, Carcinus maenas, was purified to a specific activity of 240 units/mg of protein in the assay conditions described. 2. In one method of purification the enzymic activity could be resolved into two fractions after chromatography on DEAE-cellulose. Fructose 1, 6-diphosphate was able to effect the conversion of one form (peak 1) into the second (peak 2). 3. In the presence of a saturating concentration of fructose 1, 6-diphosphate both forms of the enzyme were kinetically similar. 4. Polyacrylamide-gel electrophoresis of the enzyme 1 day after preparation showed a single protein band. On storage at least three protein bands became visible, all of which were associated with pyruvate kinase activity. 5. Chromatography of the enzyme on Sephadex G-200 indicated a mol.wt. of 247000, but in the presence of fructose 1, 6-diphosphate the elution volume of the enzyme increased corresponding to a mol.wt. of 193000. 6 Dissociation of the enzyme in sodium dodecyl sulphate and 2-mercaptoethanol followed by polyacrylamide-gel electrophoresis produced one major protein band with a mol.wt. of 55000.
Chemistry of the collagen cross-links. Nature of the cross-links in the polymorphic forms of dermal collagen during development
Authors: Bailey A J , Sims T J
Published: August 2015

Both the type I and type III collagens present in embryonic dermis are stabilized by the intermolecular cross-link, hydroxylysino-5-oxonorleucine, derived from hydroxylysine-aldehyde, although the type I collagen possesses a significant proportion of dehydrohydroxylysinonorleucine. However, concurrent with the change in the proportion of the two types of collagen during postnatal development there is a change-over with both type I and III collagens to the labile cross-link, dehydrohydroxylysinonorleucine, derived from lysine aldehyde. The results indicate that the change in the nature of the cross-link with development is determined primarily by the change in the extent of hydroxylation of the lysine residues in the terminal non-helical regions rather than being due to the change in the type of collagen.
γ-glutamyltransferase of rat kidney. Simultaneous assay of the hydrolysis and transfer reactions with (glutamate-14C)glutathione
Authors: Elce J S , Broxmeyer B
Published: August 2015

1. The hydrolytic and transfer reactions catalysed by rat kidney-gamma-glutamyltransferase (EC 2.3.2.2) were studied in vitro with substrates [U-14C]glutamic acid-labelled glutathione and methionine. Initial-velocity patterns, isotope-exchange and binding studies were consistent with a branched non-sequential mechanism in which a γ-glutamyl-enzyme intermediate may react either with water (hydrolysis) or with methionine (γ-glutamyl transfer). 2. The Michaelis constant for glutathione in hydrolysis was 13.9 +/- 1.4 μm, for glutathione in transfer it was 113 +/- 15 μM and for methionine as substrate it was 4.7 +/- 0.7 mM. At substrate concentrations in the ranges of their respective Michaelis constants, the rate of transfer was about ten times higher than that of hydrolysis, but at concentrations of methionine approximating to the physiological (64 μM in rat plasma) the transfer is negligible. 3. The enzyme is reported to lie on the luminal surface of the proximal straight kidney tubule. In this situation, if the kinetic results obtained with the detergent-solubilized enzyme are relevant to the behavior of the enzyme in vivo, it appears likely that the main function of renal γ-glutamyltransferase is not in amino acid transport, but rather to hydrolyse glutathione in the renal filtrate.
Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes
Authors: Koj A , Chudzik J , Dubin A
Published: August 2015

Two proteinases (2A and 2B) purified from the granular fraction of horse blood leucocytes degrade casein (Km values 12.8 and 6mg/ml respectively) with maximum activity at pH 7.4 and in the presence of 2m-urea. Urea-denatured haemoglobin, fibrinogen, albumin and resorcin/fuchsin-stained elastin are digested at a slower rate. The enzymes hydrolyse synthetic substrates of elastase, N-benzyloxycarbonyl-L-alanine 4-nitrophenyl ester (Km 0.114 and 0.178 mM) and N-acetyl-tri-L-alanine methyl ester (Km 5.55 and 0.98 mM), but they do not hydrolyse synthetic substrates of trypsin, chymotrypsin and thrombin. The examined proteinases are completely inhibited by 2 mM-di-isopropyl phosphorfluoridate and show a sensitivity to butyl and octyl isocyanates similar to that of pancreatic elastase. The pH-dependence of their photoinactivation in the presence of Rose Bengal indicates the presence of histidine in the active centre. Proteinase 2A rather insensitive to iodination by IC1 as is pancreatic elastase, whereas proteinase 2B is totally inactivated after incorporation of five iodine atoms per enzyme molecule.
The nucleotide sequence of cysteine transfer ribonucleic acid from baker's yeast. Identification of the products from partial degradation of the molecule and derivation of the complete sequence
Authors: Holness N J , Atfield G
Published: August 2015

1. A series of large oligonucleotide fragments derived from tRNA Cys, were separated chromatographically and the sequence of each was deduced by examination of the products of digestion with pancreatic and T1 ribonucleases. 2. The location of the specific cleavage points in the nucleotide chain was similar to that produced by brief treatment with pancreatic ribonuclease. 3. The fragments could be arranged into two alternative sequences. The correct sequence was deduced by the sequential removal and identification of the first nine nucleotides from the 3′-end of the terminal half of the molecules.
The structure of haem in pyridine/water mixtures and its implication in studies of haem catabolism
Authors: Brown S B , King R F G J
Published: August 2015

1. A study of haem spectra in pyridine/water mixtures at low pyridine concentrations revealed changes in haemochrome structure consistent with an aggregation process. No corresponding change in the structure of the haemichrome species was observed. 2. This aggregation has been correlated with a previously observed sharp decrease in the rate of coupled oxidation (degradation) of haem as pyridine concentration is decreased. The decrease appears to be due primarily to haem aggregation and not to changes in the hydrophobic nature of the solvent. The effect of ethanol and butanone addition was examined and supports this conclusion. 3. Evidence is presented that coupled oxidation occurs via the iron (II) species (haemochrome).
Inhibition of spinach phosphoribulokinase by DL-glyceraldehyde
Authors: Slabas A R , Walker D A
Published: August 2015

Spinach chloroplast phosphoribulokinase is inhibited by DL-glyceraldehyde. The inhibition is non-competitive with respect to ribulose 5-phosphate (Ki 19mM) and ATP (Ki 20mM). The inhibition is discussed in relation to a previously reported inhibition of CO2 assimilation in intact and envelope-free chloroplasts by DL-glyceraldehyde. It is concluded that the inhibition of phosphoribulokinase is insufficient to account for the inhibition, by DL-glyceraldehyde, of O2 evolution with ribose 5-phosphate as substrate and that a further site of inhibition is also present in this system.
Formation of monoferric ovotransferrins in the presence of chelates
Authors: Donovan J W , Beardslee R A , Ross K D
Published: August 2015

1. When ovotransferrin is partially saturated with iron, endotherms for apo-ovotransferrin, two monoferric ovotransferrins and Fe2-ovotransferrin are observed by differential scanning calorimetry. The relative sizes of the endotherms are changed in the presence of the iron-chelating agents nitrilotriacetic acid and ATP. 2. When iron is added as Fe(III)-nitrilotriacetate, at Fe-nitrilotriacetate: ovotransferrin ratios less than unity, the endotherm for Fe2-ovotransferrin is essentially absent. At Fe-nitrilotriacetate: ovotransferrin ratios of unity the only species present in solution in appreciable concentration as evidenced by their differential-scanning-calorimetry endotherms, are two monoferric ovotransferrins in approximately equal amounts. At Fe-nitrilotriacetate: ovotransferrin ratios greater than unity, the apo-ovotransferrin endotherm is absent, and the endotherms for the two monoferric ovotransferrins decrease in size as the endotherm for Fe2-ovotransferrin increases. 3. In the presence of nitrilotriacetate, binding of iron to the two sites of ovotransferrin is highly anti-co-operative, but essentially indiscriminate. When monoferric ovotransferrin is formed from apo-ovotransferrin, binding at one site is slightly favoured compared with binding at the other site, but once iron has been bound at either site, the binding affinity for iron at the unoccupied site is much decreased.
Changes in the activities of the enzymes of hepatic fatty acid oxidation during development of the rat
Authors: Foster P C , Bailey E
Published: August 2015

1. Changes in the activities of several enzymes involved in mitochondrial fatty acid oxidation were measured in livers of developing rats between late foetal life and maturity. The enzymes studied are medium- and long-chain ATP-dependent acyl-CoA synthetases of the outer mitochondrial membrane and matrix, GTP-dependent acyl-CoA synthetase, carnitine acyltransferase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, general 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase.
The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex
Authors: Baker R R , Dowdall M J , Whittaker V P
Published: August 2015

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.
Role of oxygen on growth rate and gene activity in cultured chick-embryo heart cells
Authors: Clo' C , Orlandini G C , Guarnieri C , Caldarera C M
Published: August 2015

Environmental oxygen is shown to have a regulatory role in growth rate and the mechanism of gene de-repression in chick-embryo heart cells; it modulates the intracellular concentration of polyamines, and this appears to be closely related to histone acetylation.
Accumulation and turnover of the classical Folch-Lees proteolipid proteins in developing and adult rat brain
Authors: Agrawal H C , Fujimoto K , Burton R M
Published: August 2015

The turnover of classical Folch-Lees proteolipid proteins was studied after administration of [2,3-3H]tryptophan to both developing and adult rat brain. The animals were killed from 2h to 250 days after subcutaneous injections of [3H]tryptophan. The measured specific radioactivity in developing brain attained maximum value 24h after the administration of label, whereas the total radioactivity per brain reached a maximum 21 days after injection. The half-life of proteolipid protein from the measured specific radioactivity was 7-20 days, depending on the time-points used for the calculation, whereas calculation from total radioactivity between 28-77 and 91-257 days gave half-lives of 35-40 and 188 days respectively. In contrast, in animals injected at 40 days of age, the half-life from the whole-brain-radioactivity data was 188 days. The problem of the recycling of radioactivity for the synthesis of myelin proteins from either a general or a discrete amino acid pool is discussed.
Nucleo-cytoplasmic relationships of high-molecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain
Authors: Berthold W , Lim L
Published: August 2015

The metabolism of high-molecular-weight RNA in the nuclear and cytoplasmic fractions of newborn and adult rat brain was investigated after the intracranial administration of [32P]Pi. In young brain, a considerable proportion of the newly synthesized radioactive RNA is transferred to the cytoplasm, in contrast with the adult brain, where there appears to be a high intranuclear turnover. Electrophoretic analysis of the newly synthesized RNA showed that processing of the rRNA precursor to yield the 28S and 18S rRNA may be more rapid in the adult than in the young, although most of the adult rRNA in the nucleus is not transferred to the cytoplasm. In young brain, processing is probably tightly coupled to transport of rRNA into the cytoplasm, so that 28S and 18S rRNA are not subjected to possible degradation within the nucleus. Polyadenylated RNA turns over in concert with high-molecular-weight RNA in the nuclei of the adult rat brain. In the cytoplasm the polyadenylated RNA has a higher turnover rate relative to rRNA. In the young brain the polyadenylated RNA is transferred to the cytoplasm along with rRNA, although polyadenylated RNA is transported into the cytoplasm at a faster rate. The nuclear and cytoplasmic polyadenylated RNA species of young brain are larger than their corresponding adult counterparts. These results suggest that there are considerable changes in the regulation of the nucleo-cytoplasmic relationship of rRNA and polyadenylated RNA during the transition of the brain from a developing replicative phase to an adult differentiated and non-dividing state.
The regulation of phenylalanine hydroxylase in rat tissues in vivo. Substrate- and cortisol-induced elevations in phenylalanine hydroxylase activity
Authors: Greengard O , Delvalle J A.
Published: August 2015

Injections of phenylalanine increased a 2.5-fold in 9 h the hepatic phenylalanine hydroxylase activity of 6-day-old or adult rats that had been pretreated (24h earlier) with p-chlorophenylalanine; without such pretreatment, phenylalanine did not raise the enzyme concentration. This difference is paralleled by the much greater extent to which the injected phenylalanine accumulated in livers of the pretreated compared with the normal animals. The hormonal induction of hepatic phenylalanine hydroxylase activity obeyed different rules: an injection of cortisol was without effect on adult livers but caused a threefold rise in phenylalanine hydroxylase activity of immature ones, both without and after pretreatment with p-chlorophenylalanine. In the latter instance, the effects of cortisol, and of phenylalanine were additive. Actinomycin inhibited the cortisol- but not the substrate-induced increase of phenylalanine hydroxylase, whereas puromycin inhibited both. The results indicate that substrate and hormone, two potential positive regulators of the amount of the hepatic (but not the renal) phenylalanine hydroxylase, act independently by two different mechanisms. The negative effector, p-chlorophenylalanine, also appears to interact with the synthetic (or degradative) machinery rather than with the existing phenylalanine hydroxylase molecules: 24h were required in vivo for an 85% decrease to ensue, and no inhibition occurred in vitro when incubating the enzyme with p-chlorophenylalanine or with liver extracts from p-chlorophenylalanine-treated rats.
Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315
Authors: Dwek R A , Givol D , Jones R , McLaughlin A C , Wain-Hobson S , White A I , Wright C
Published: August 2015

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a ‘slow’ conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment.
The mechanism of sensitivity to phleomycin in growing Escherichia coli cells
Authors: Sleigh M J , Grigg G W
Published: August 2015

Stationary-phase Escherichia coli B cells transferred to new growth medium are initially resistant to net DNA breakage by low concentrations of phleomycin, and become sensitive as DNA replication commences. From studies with inhibitors of various stages of the DNA replication cycle it is evident that it is not DNA synthesis itself that is required for induction of DNA breakage by phleomycin, but events associated with the initiation of DNA replication. Termination of replication in the absence of further initiaiton results in resistance to phleomycin. The cellular change responsible for changes in sensitivity to phleomycin could be the attachment of the bacterial chromosome to the cell membrane at initiation and detachment on termination of replication, suggesting an alteration in the balance between cellular DNA breakage and repair processes for membrane-associated compared with non-membrane-associated DNA.
Partial purification and characterization of collagen galactosyltransferase from chick embryos
Authors: Risteli L , Myllyä R , Kivirikko K I
Published: August 2015

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.
Isolation and properties of α-D-mannosidase from human kidney
Authors: Marinkovic D V. , Marinkovic J N.
Published: August 2015

α-D-Mannosidase activity exists in three forms that can be separated by DEAE-cellulose chromatography, α-D-Mannosidase was isolated from human kidney in a homogeneous state, and was purified 2100-fold, with p-nitrophenyl α-D-mannoside as substrate. The purified α-D-mannosidase was practically free from all other glycosidases tested. The Km of the synthetic substrate with the enzyme was 1 × 10(-3) M and the pH optimum 4.5. It was inhibited by heavy metals, sodium dodecyl sulphate, urea and compounds that react with the thiol groups, and was activated by Zn2+, Na+, 2-mercaptoethanol, human albumin and γ-globulin. The mol. wt. of the enzyme was estimated to be 180 000 +/- 4500. After pretreatment with 2-mercaptoethanol and sodium dodecyl sulphate, α-D-mannosidase dissociated into subunits of mol. wts. of 58 000 +/- 600 and 30 000 +/- 380 respectively. Subunits of the same molecular weights were also obtained after the enzyme was heated at 100 degrees C.
Kinetic studies on the binding of cyanide to oxygenated cytochrome c oxidase
Authors: Brittain T , Greenwood C
Published: August 2015

The reaction of cyanide with oxygenated cytochrome c oxidase was followed by means of flow-flash techniques. The oxygenated form, produced after photolysis of the partially reduced CO complex in the presence of cyanide and O2, shows cyanide-binding properties distinct from those of both the oxidized and the reduced forms of the protein. The binding is a single process (k = 22M-1-S-1) linearly dependent on cyanide concentration to as high as 75 mM. It is suggested that the oxygenated form is a conformational variant of the oxidized protein.
The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins
Authors: Chaplin M F.
Published: August 2015

A simple technique was developed for the specific reversible modification of guanidino groups in proteins involving reaction with ninhydrin. The extent of the reaction is easily determined non-destructively by spectrophotometric analysis. The reagent can also be used for the titration of sterically unhindered thiol groups in proteins.
Spectroscopic evidence for the uneven distribution of adenine and uracil residues in ribosomal ribonucleic acid of Drosophila melanogaster and of Plasmodium knowlesi and its possible evolutionary significance
Authors: Cox R A , Godwin E , Hastings J R
Published: August 2015

RNA was isolated from subribosomal particles of the malaria parasite Plasmodium knowlesi. The nucleotide composition (mole fraction) of the principal species was obtained (S-rRNA, 0.295A, 0.36U, 0.25G, 0.105C: L-rRNA, 0.326A, 0.31U, 0.228G, 0.144C). Ribosomal RNA was also isolated from Drosophila melanogaster. Optical properties of these A + U-rich species were measured. In all four cases analysis of the hypochromic effect revealed that adenine and uracil residues tended to form clusters along the polynucleotide chain. A substantial fraction of residues was located in bihelical regions of approx. 50% G-C base pairs or in regions of approx. 30-35% G-C base pairs. The possible evolutionary significance of these results was considered on the basis of comparison with properties of rRNA from bacteria (Escherichia coli) and a mammal (rabbit reticulocyte).
A new method for deriving steady-state rate equations suitable for manual or computer use
Authors: Indge K J , Childs R E
Published: August 2015

A schematic method for the derivation of steady-state enzyme rate equations by using the Wang algebra is described. The method is simple, easy to learn and offers a substantial decrease in analytical effort over previously published algorithms. Being essentially an algebraic procedure the method can be readily computerized. Computer programs in BASIC and ALGOL languages have been deposited as Supplementary Publication SUP 50065 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1976). 153, 5.
Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa
Authors: Flanagan P R , Zbarsky S H
Published: August 2015

Phosphodiesterase II from extracts of intestinal mucosa of rat and guinea pig was purified by chromatography on DEAE-cellulose, CM-cellulose and agarose. The rat enzyme was purified 350-550-fold, with recoveries ranging up to 46%. The best purification of the guinea-pig enzyme was 15-fold, and the recovery was only 2.6%, the large loss occurring during chromatography on DEAE-cellulose and agarose. The poor results with the guinea-pig enzyme reflect the difficulty in obtaining a truly soluble material. Repeated sonication of the crude guinea-pig preparations yielded material that was initially soluble but tended to re-aggregate quickly. Purification of the rat phosphodiesterase II increased its thermostability, the temperature of half-inactivation being increased from 54degrees to 60degreesC. Both enzymes had a Km value of 4 × 10(-5) M with thymidine 3′-(2,4-dinitrophenyl) phosphate as substrate and showed similar pH optima for activity. Both enzymes were inhibited slightly in 0.1 M-MgC12 or 2M-urea and much more strongly in 2M-(NH4)2SO4 or 6M-NaC1. The guinea-pig enzyme was usually inhibited more than the rat enzyme. The Arrhenius plots of the two enzymes differed slightly in slope, but both were biphasic, showing breaks between 30degrees and 40degreesC. It was concluded that the two enzymes were markedly similar in behaviour and that the differences found were related to the different degrees of purification attained by the procedures described.
Mechanism of increased hepatic uptake of unesterified fatty acid from serum of ethanol-treated rats
Authors: Abrams M A , Cooper C
Published: August 2015

Studies were made on the mechanism by which livers of ethanol-treated rats take up an increased fraction of the total flux of unesterified fatty acid in serum. It was found that ethanol (0.7g/kg) causes a twofold rise in the serum content of liver, and that this serum is in rapid equilibrium with the general circulation. The fractional hepatic uptake from serum of group of compounds with varying uptake mechanisms and metabolic fates was studied in control and ethanol-treated animals. All the compounds tested, including unesterified fatty acid, showed an enhanced uptake when ethanol was given. For one of the compounds, carbon tetrachloride, a dose/response relationship was established between the amount administered, the amount taken up by liver, and the amount metabolized. These findings were interpreted to mean that this dose of ethanol causes the liver to receive an increased flow of blood, and as a result all compounds present and capable of being taken by liver are taken up at an increased rate. Hepatic blood flow was measured by a technique that monitors the rate of clearance of a colloidal lipid emulsion. It was found that ethanol increased hepatic blood flow by about 60%. This effect of ethanol on hepatic blood flow provides an explanation for the fatty liver and the synergistic effect between an acute dose of ethanol and carbon tetrachloride. A hypothesis to explain why a moderate dose of ethanol causes triglyceride to accumulate in liver is presented.
Nuclear components responsible for the retention of steroid–receptor complexes, especially from the standpoint of the specifcity of hormonal responses
Authors: Mainwaring W I , Symes E K , Higgins S J
Published: August 2015

1. By covalently linking nuclear components from hormone-sensitive cells to Sepharose 2B, it is possible to investigate the interaction between nuclear components and cytoplasmic receptor-steroid complexes by affinity chromatography. 2. Many factors are implicated in the specifity of nuclear-cytoplasmic interactions, including the nature of the nuclear components, the presence of the cytoplasmic receptor protein and the provision of the appropriate steroid ligand. 3. Two distinct sets of binding sites are present in nuclear extracts immobilized to Sepharose 2B, namely a small number of specific high-affinity sites and a larger number of non-specific low affinity-sites. 4. Considerable evidence supports the importance of the high-affinity binding sites in the manifestation of hormonal specificity in different tissues. Although the study has centred largely on androgenresponsive systems, the findings are germane to cytoplasmic-nuclear interactions in general. 5. The high-affinity or acceptor sites in nuclear extracts reside in the basic but non-histone protein fraction. 6. Hormonal specificity is seemingly maintained by both the cytoplasmic and nuclear components, and the results are discussed in the context of the mechanism of action of steroid hormones.
Rates of induction of mitochondrial aldehyde dehydrogenase in rat liver
Authors: Horton A A , Barrett M C
Published: August 2015

Aldehyde dehydrogenase activities in liver mitochondria isolated from rats given ethanol at hourly intervals by gastric intubation showed a brief lag period followed by a rapid increase in specific activities until a maximum was attained at about 3h.
16β-hydroxylation of dehydroepiandrosterone sulphate by homogenates of human foetal liver
Authors: Wynne K N , Renwick A G C
Published: August 2015

The chemical syntheses of the 3-sulphates of 16 α-acetoxy-, 16 α-hydroxy- and 16 β-hydroxy-dehydroepiandrosterone as their triethylammonium salts are described preparatory to studies of the metabolism of [7 α-3 H]dehydroepiandrosterone sulphate by homogenates of human foetal liver. Five main radioactive products of the incubation were separated by partition chromatography on a Celite column. Two were identified as 16 α-and 16 β-hydroxydehydroepiandrosterone 3-sulphates by crystallization to constant specific radioactivity before and after solvolysis. The yields of the conversion to the two epimers were 24.4% (16 α) and 1.8% (16 beta).
The existence of a glyconeogenic pathway in rat skin
Authors: Peters R F , White A M
Published: August 2015

The existence of a glyconeogenic pathway in rat skin has been demonstrated by measurement of three of the key glyconeogenic enzymes, fructose 1,6-bisphosphatase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase, and by studies on the incorporation in vitro of carbon from pyruvate and alanine into skin glycogen.
Glucose transport as rate-limiting step in the growth of Escherichia coli on glucose
Authors: Herbert D , Kornberg H L
Published: August 2015

Over a wide range of growth rates, two strains of Escherichia coli growing aerobically in continuous culture under glucose limitation utilized glucose at rates identical with those at which cells harvested from the chemostats transported [14C]glucose.
The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells
Authors: Aujame L , Freeman K B
Published: August 2015

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.
Fatty acid synthesis in the perfused liver of adrenalectomized rats
Authors: Kirk C J , Verrinder T R , Hems D A
Published: August 2015

1. Fatty acid synthesis, measured in the perfused liver of fed adrenalectomized rats with 3H2O and 14C-labelled precursors, was less than in control sham-operated rats. 2. This defect was more extensive for synthesis of fatty acids incorporated into triacylglycerols than into phospholipids. 3. There was impairment in desaturation and export of newly synthesized fatty acid. 4. Fatty acid synthesis and desaturation were restored to normal rates 5h after treatment with cortisol in vivo. 5. Fatty acid synthesis was seasonally variable, being highest in the winter; the impairment after adrenalectomy was observed in all seasons. 6. In perfusions with oleate (0.7 mM), no further impairment in fatty acid synthesis was discerned in livers from adrenalectomized rats, in which the rate resembled that in control livers. 7. No defect in the incorporation of oleate into glycerides was discerned in livers from adrenalectomized rats. 8. Cortisol exerted no stimulatory effect on fatty acid synthesis when added to perfusion media. 9. The impairment in hepatic lipogenesis, demonstrable after adrenalectomy, shows that adrenal glucocorticoids promote hepatic capacity for fatty acid synthesis de novo, at least in intact non-diabetic rats. It is suggested that this effect is mediated by insulin, perhaps through direct action on the liver.
Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate
Authors: Clark M G , Filsell O H , Jarrett I G
Published: August 2015

1. Isolated lamb liver cells were prepared from 24-h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase. On the basis of Trypan-Blue exclusion, rate of O2 uptake, adenine nucleotide content and retention of constitutive enzymes, these cells were judged to be intact. 2. Isolated caudate-lobe liver cells showed rates of gluconeogenesis from 10 mM-propionate and 10 mM-lactate that compared favourably with rates determined in isolated median-lobe cells and with rates determined with the isolated perfused lamb liver. 3. The gluconeogenic potential of substrates tested depended on the lamb's age. Cells prepared from suckling lambs (up to 20 days of age and essentially non-ruminant) showed highest rates from galactose, serine and alanine; those prepared from post-weaned lambs (older than 30 days of age and ruminant) showed highest rates from propionate, lactate and fructose. 4. Gluconeogenic rates from endogeneous precursors, 10 mM-propionate and 10mM-galactose, were linear for 1 h and were both stimulated by 1 muM-glucagon. Provided the endogenous rate of gluconeogenesis remained unchanged after substrate addition, glucagon caused a net stimulation of gluconeogenesis from each of these substrates. 5. Gluconeogenic capacity and glucagon sensitivity were examined in cells maintained in substrate-free oxygenated buffer at 37 degrees, 22 degrees and * degrees C. Even under the best of the three conditions of storage that were tested (i.e. at 22 degrees C in gelatin-containing buffer) deterioration of the lamb cells proceeded rapidly, and loss of glucagon responsiveness preceeded the loss of ability to convert precursor into glucose. 6. n-Butyric acid, 2-methylpropanoic acid and 3-methylbutanoic acid at concentrations comparable with those found in lamb portal-vein blood each stimulated gluconeogenesis from 10mM-galactose or 10mM-propionate; gluconeogenesis from galactose was stimulated to the greater extent. 7. The regulatory effects of glucagon and sodium butyrate on lamb liver-cell gluconeogenesis and glycogenolysis were compared. Glucagon (1 muM) and 2mM-butyrate accelerated the rate of glucose formation of liver cells of 24h-starved animals from lactate+pyruvate or fructose. Insulin (20nM) decreased both gluconeogenesis and the efficacy of 1 muM-glucagon. For lactate+pyruvate as substrate, the stimulatory effect of butyrate was additive to that of 1muM-glucagon and for both lactate+pyruvate and fructose the stimulatory effect of butyrate was not influenced by 20nM-insulin. In contrast with glucagon, which stimulated the rate of glycogenolysis in cells prepared from fed lambs, butyrate (0.1-20mM) had no effect. 8. It is concluded that glucagon and butyrate stimulate lamb liver-cell gluconeogenesis by different mechanisms.
Elongation of [ω-14C]oleic acid and [ω-14C]nervonic acid
Authors: Rabinowitz J L , Lutton C , Chevallier F
Published: August 2015

During feeding experiments with [ω-14C]oleic acid and [ω-14c]nervonic acid to adult rats, 14C-labelled C26, C28 and C30 fatty acids were recovered from the intestinal mucosa, liver, plasma, kidney and stools. The structures of these fatty acids were determined by g.l.c., radio-g.l.c. and mass spectrometry. The Schmidt and Ginger degradation methods indicated that most of the 14C found in these extra-long fatty acids remained in the omega position. These radioactive extra-long fatty acids were found mainly in the polar lipids of rats killed 3 or 15 h after being fed on labelled oleic acid or nervonic acid. Rats killed 63 h later yielded only traces of these extra-long fatty acids. When the rats were given antibiotics or received the same radioactive fatty acids by intravenous injection, the labelled extra-long fatty acids could not be detected in any of the tissues. We conclude that they were probably synthesized by elongation of oleic acid and nervonic acid by intestinal micro-organisms (probably yeasts) and then absorbed by the intestinal mucosa.
An explanation of apparent sudden change in the activation energy of membrane enzymes
Authors: Wynn-Williams A T
Published: August 2015

If an enzyme-lipid mixture forms phases of pure lipid and enzyme-lipid solution, and enzyme activity depends on the composition of the enzyme-lipid solution, the temperature-dependence of lipid solubility in the enzyme-lipid solution leads to apparent sudden changes in enzyme activation energy without activity discontinuities at lipid phase transition temperatures.
The 3H-labelling of bilirubin
Authors: Hancock F E , Hutchinson D W , Knell A J
Published: August 2015

A simple method has been developed for the preparation of 3H-labelled bilirubin IX-alpha from bilirubin dimethyl ester. The label is located in the propionic acid side chains, and there is no isomerization of the bilirubin during the preparation.
Mitochondrial glycerol kinase activity in rat brain
Authors: Tildon J T , Stevenson J H , Ozand P T
Published: August 2015

Glycerol kinase activity was found in the particulate fraction of rat brain homogenates predominantly associated with mitochondria. The enzyme remained bound to the particulate fraction after treatment with a variety of solubilizing agents.
Absence of binding of pancreatic and urinary kallikreins to α 2-macroglobulin
Authors: Vahtera E , Hamberg U
Published: August 2015

Pancreatic and urinary kallikreins failed to form the typical serine proteinase complex with alpha2M (alpha2-macroglobulin). Studies were performed to compare this with the binding of trypsin to alpha2M at various molar binding ratios, with the use of Sephadex G-200 gel filtration to separate free and alpha2M-bound enzyme fractions. The subunit conversion was totally absent with pancreatic kallikrein from lhich traces of a binding proteinase had been removed. The lack of binding is believed to be the result of the restricted specificity of the kallikreins.
The kinetics of rabbit muscle pyruvate kinase. Initial-velocity, substrate- and product-inhibition and isotopic-exchange studies of the reverse reaction
Authors: Giles I G , Poat P C , Munday K A
Published: August 2015

1. An assay, based on the transfer of label from [gamma-32P]ATP to [32P]phosphoenolpyruvate, suitable for a steady-state kinetic analysis of pyruvate kinase in the reverse direction (i.e. phosphoenolpyruvate synthesis), is described. 2. This assay was used in a kinetic investigation of the rabbit muscle enzyme including initial-rate and product-inhibition experiments, at a pH of 7.4 and constant concentrations of total K+ and free Mg2+. 3. These studies indicate that there is a random release of ADP and phosphoenolpyruvate from the enzyme and that there is a competitive substrate inhibition by ATP. Some of the results were suggestive that the rapid-equilibrium assumption, generally used for this enzyme was not valid. 4. Techniques were developed to measure the rate of isotopic exchange between all the substrate-product pairs. 5. By using these techniques the rates of isotopic exchange at chemical equilibrium were measured. The results indicate that this enzyme does not catalyse a truly rapid-equilibrium random mechanism, although in the forward reaction all initial-rate data obtained to date are consistent with this assumption.