bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of bone matrix and tendon with particular reference to glycoprotein content
Authors: Herring G M
Published: August 2015

Bone matrix and tendon are compared in terms of their carbohydrate and non-collagenous protein composition. The collagen content of both tissues was similar (90-91%), but bone matrix had at least three times as much sialic acid (0.28%) as tendon (0.08%). Smaller differences were found in the analysis of hexoses and hexosamines. After digestion with bacterial collagenase, about 9% of the total protein from both tissues was non-diffusible on dialysis, and this contained only 0.15% (bone) and 0.7% (tendon) of the original hydroxyproline; recovery of sialic acid was 86-87%. The collagenase-resistant soluble material amounted to about 9% (bone matrix) and 5% (tendon); the insoluble residues were 1 and 4% respectively. There were clear differences in the carbohydrate contents of the digests, but the amino acid compositions were similar. When the soluble digests were chromatographed on DEAE-cellulose, the elution profiles indicated the presence in each tissue of a variety of glycoproteins and a proteoglycan fraction, and showed clearly that an acidic glycoprotein corresponding to bone sialoprotein was not present in tendon.
Trypsin-induced phospholipase activity in human platelets
Authors: Pickett W C , Jesse R L , Cohen P
Published: August 2015

Trypsin mediates a release of arachidonic acid with resultant increase in O2 consumption (a reflection of cyclo-oxygenase activity) by whole human platelets that is similar to thrombin's effect on these cells. The trypsin and thrombin effects can be differentiated in two ways: (1) at saturating concentrations the measured effects of trypsin greatly exceed those of thrombin; (2) EGTA [ethanedioxybis(ethylamine)-NNN′N′-tera-acetate] augments the effect of thrombin but not of trypsin. Thus trypsin and thrombin probably act at different loci in the pathway that induces phospholipase activity in human platelets.
Total plasmalogens and O-(acylalkylglycerophosphoryl) ethanolamine from labelled hexadecanol and palmitate during hypoxia and anoxia in rat heart
Authors: Rabinowitz J L , Hercker E S
Published: August 2015

By the use of the Langendorff technique, surviving isolated rat hearts were perfused with [1-14 C] palmitate, [1-14C] hexadecanol or [1-14C,1-3H] hexadecanol under normal or anoxic conditions. After perfusion for 30min with either precursor, when oxygenated or in an hypoxic condition, or when 1mM-KCN was included in the system, the heart tissues showed no significant chemical changes in their content of total lipids, total phospholipids or total ethanolamine-containing phospholipids. Changes were observed in the ratio of alkyl-to alk-1-enyl-glycerophosphorylethanolamine in the tissue perfused with N2+CO2 plus CN-. A slight increase from 4.0±0.3 to 4.9±0.2% in alkyl derivatives and a decrease from 11.2±0.4 to 9.4±0.3% in alk-1-enyl derivatives was observed. The incorporation of the [14C] palmitate and the [14C] hexadecanol into the recovered phospholipids and plasmalogens was severely decreased in the tissues perfused with CN-: in the hypoxic state only a mild inhibition was observed compared with the oxygenated systems. Considerable 3H from [1-14C, 1-3H] hexadecanol was retained (25-35%) in the alk-1-enylether chains of plasmalogens under both the oxygenated conditions and with CN-, suggesting that the same mechanism of incorporation is operational at high or low O2 concentrations. The results are consistent with an O2-dependent, CN-sensitive step in the biosynthesis of plasmalogens in the rat heart.
Developmental changes in purine phosphoribosyltransferases in human and rat tissues
Authors: Adams A , Harkness R A
Published: August 2015

1. The hypoxanthine/guanine and adenine phosphoribosyltransferase activities in a wide variety of human tissues were studied during their growth and development from foetal life onward. A wide range of activities develop after birth, with especially high values in the central nervous system and testes. 2. Postnatal development of hypoxanthine/guanine phosphoribosyltransferase was also defined in the rat. Although there were increases in the central nervous system and testes, there was also a rise in activity in the liver, which was less marked in man. 3. A sensitive radiochemical assay method, using dTTP to inhibit 5′-nucleotidase activity, suitable for tissue extracts, was developed. 4. No definite evidence of the existence of tissue-specific isoenzymes of hypoxanthine/guanine or adenine phosphoribosyltransferase was found. Hypoxanthine/guanine phosphoribosyltransferase in testes, however, had a significantly different thermal-denaturation rate constant. 5. The findings are discussed in an attempt to relate activity of hypoxanthine/guanine phosphoribosyltransferase to biological function. Growth as well as some developmental changes appear to be related to increase in the activity of this enzyme.
The use of gadolinium as a probe in the Fc region of a homogeneous anti-(type-III pneumococcal polysaccharide) antibody
Authors: Willan K J , Wallace K H , Jaton J C , Dwek R A
Published: August 2015

The binding of gadolinium [Gd(III)] to a homogeneous rabbit anti-(type-III pneumococcal polysaccharide) IgG (immunoglobulin G) and its Fab (N-terminal half of heavy and light chain) and Fc (C-terminal half of heavy-chain dimer) fragments was demonstrated by measurements of solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 the binding of Gd(III) to the Fc fragment is much tighter (KD approx. 5 micronM) than binding to the Fab fragment (KD approx. 250 micronM). The binding of Gd(III) to the whole IgG molecule (KD approx. 4 micronM) is very similar to that for the Fc fragment alone. This specificity of binding to the Fc region allows the use of Gd(III) as a probe of the Fc conformation. The environment of the Gd(III) in the Fc region of whole IgG is not affected by the presence of octasaccharide derived by hydrolysis of type-III pneumococcal polysaccharide, but the corresponding 28-unit saccharide does cause detectable changes. The addition of 16-unit saccharide to anti-(SIII polysaccharide) IgG in the presence of Gd(III) does not change the solvent water proton relaxation rate, although aggregation does occur. The effects of the 28-unit saccharide may be explained therefore by a change in the tumbling time of the IgG. From a study of the effect of various antigen/antibody ratios, it is concluded that the 28-unit-saccharide-induced changes in the Gd(III) environment in the Fc region are caused by the specific geometrical structure of the antigen-antibody complexes formed, and not simply by occupancy of the combining sites on the antibody.
Multiple enzyme purifications from muscle extracts by using affinity-elution-chromatographic procedures
Authors: Scopes R K
Published: August 2015

1. Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying four to six separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. 2. Schemes for purifying 12 separate enzymes from rabbit muscle, and eight from chicken muscle extracts, are included. In nearly all cases the overall procedure involves three steps: the initial (NH4)2SO4 fractionation, the ion-exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. 3. The five schemes described include illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also include stepwise adsorption on columns at different pH values. 4. Separation of two electrophoretically differing forms of phosphoglycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form.
Improved non-parametric statistical methods for the estimation of Michaelis-Menten kinetic parameters by the direct linear plot
Authors: Porter W R , Trager W F
Published: August 2015

The theoretical basis for the direct linear plot [Eisenthal & Cornish-Bowden (1974) Biochem. J. 139, 715-720], a non-parametric statistical method for the analysis of data-fitting the Michaelis-Menten equation, was reinvestigated in order to accommodate additional experimental designs and to provide estimates of precision more directly comparable with those obtained by parametric statistical methods. Methods are given for calculating upper and lower confidence limits for the estimated parameters, for accommodating replicate measurements and for comparing the results of two separate experiments. Factors that influence the proper design of experiments are discussed.
A new amino acid, 3-(2,5-SS-dicysteinyl-3,4-dihydroxyphenyl)alanine, from the tapetum lucidum of the gar (Lepisosteidae) and its enzymic synthesis
Authors: Ito S , Nicol J A C
Published: August 2015

The tapetum lucidum of the alligator gar Lepisosteus was shown by t.l.c. to contain a new phenolic amino acid, which is apparently a major constituent of the reflecting material. It was isolated in a yield of 0.5 mg/eye and its physical and chemical characteristics, especially reductive hydrolysis with hydriodic acid giving dopa (3,4-dihydroxyphenylalanine) and cysteine, suggested that it might to SS-dicysteinyldopa. Tyrosinase oxidation of L-dopa in the presence of an excess of L-cysteine yielded, in addition to known 5- and 2-S-cysteinyldopa, the same amino acid as that isolated from the eye of the gar, thus confirming the gross structure. The position of the two cysteine residues was established by the fact that tyrosinase oxidation of catechol and cyteine gave 3-S-cysteinylcatechol and 3,6-SS-dicysteinylcatechol. The natural amino acid is therefore formulated as 3-(2,5-SS-dicysteinyl-3,4-dihydroxyphenyl)alanine (2,5-SS-dicysteinyldopa), which may be formed by two consecutive additions of cysteine, first to dopaquinone and then to 5-S-cysteinyldopaquinone. The enzymic synthesis of 2,5-SS-dicysteinyldopa in vitro suggests that it may also be involved in the biosynthesis of phaeomelanin.
Differences in catalytic properties between cerebral cytoplasmic and mitochondrial hexokinases
Authors: Thompson M F , Bachelard H S
Published: August 2015

1. Clear kinetic differences between cytoplasmic and mitochondrial forms of type-I cerebral hexokinase were demonstrated from experiments performed under identical conditions on three (cytoplasmic, bound mitochondrial and solubilized mitochondrial) preparations of the enzyme. 2. Whereas the Michaelis constant for glucose (KmGlc) was consistent, that for MgATP2- (KmATP) was lower in the cytoplasmic than in the two mitochondrial preparations. The substrate dissociation constants (KsGlc and KsATP) were both higher in the cytoplasmic than in the mitochondrial preparations. A further difference in the substrate kinetic patterns was that KmATP=KmATP for the cytoplasmic enzyme, in contrast with the mitochondrial enzyme, where KmATP was clearly not equal to KsATP [Bachelard et al. (1971) Biochem. J. 123, 707-715]. 3. Dead-end inhibition produced by N-acetyl-glucosamine and by AMP also exhibited different quantitative kinetic patterns for the two enzyme sources. Both inhibitions gave Ki values similar or equal to those of Ki′ for the cytoplasmic activity, whereas Ki was clearly not equal to Ki′ for the mitochondrial activity. 4. All of these studies demonstrated the similarity of the two mitochondrial activities (particulate and solubilized), which were both clearly different from the cytoplasmic activity. 5. The analysis gives a practical example of our previous theoretical treatment on the derivation of true inhibition constants. 6. The results are discussed in terms of the function of cerebral hexokinases.
Cholera toxin requires oxidized nicotinamide-adenine dinucleotide to activate adenylate cyclase in purified rat liver plasma membranes
Authors: Martin B R , Houslay M D , Kennedy E L
Published: August 2015

Activation of adenylate cyclase in isolated rat liver plasma membranes by cholera toxin was demonstrated. The activation requires the presence of NAD+ and ATP and is irreversible.
The turnover of hamster fibroblast lysosomal β-d-glucuronidase
Authors: Warburton M J , Wynn C H
Published: August 2015

The half-life of hamster fibroblast beta-D-glucuronidase (EC 3.2.1.31) was estimated to be 4-5 days by measuring the decay with time of the radioactivity in beta-D-glucuronidase isolated from cells grown in the presence of 14C-labelled amino acids. A new affinity-chromatographic procedure for the purification of beta-D-glucuronidase is described.
Protein determinants of myelination in different regions of developing rat central nervous system
Authors: Banik N L , Smith M E
Published: August 2015

Measurements of several different protein determinants correlated with the time and rate of myelination in five areas of the central nervous system are presented. The deposition of protein in the subcellular fraction corresponding to the density of adult myelin, the appearance of basic protein characteristic myelin, the change in proportions of the individual myelin proteins, the appearance and distribution of the myelin marker 2′:3′-cyclic nucleotide3′-phosphohydrolase, and the results of morphological studies of purified myelin are compared. According to these various criteria, and in agreement with the morphological observations of others, myelin appears earliest in the spinal cord, then in the brain stem, and latest in the cerebral hemispheres. Multilamellar myelin was observed in the rat brain stem and spinal cord as early as 5 days of age. The relative proportion of the individual myelin proteins changed with myelin maturation in all areas, with the larger basic protein decreasing reciprocally with increase of the smaller basic protein. The proportion of Wolfgram protein also decreased with maturation. Larger proportions of the enzyme 2′:3′-cyclic nucleotide 3′-phosphohydrolase were located in the microsomal fraction at early ages. During development the enzyme activity gradually became associated more with a fraction of a density corresponding to adult myelin, suggesting the presence of precursor membrane fragments in microsomal fractions in the early stages of myelination before compact myelin formation. A significant proportion of the total nucleotide phosphohydrolase activity of the homogenate could not be recovered in subcellular fraction at early ages, but the recovers of the enzyme increased with maturation and the activity was found more in the myelin fraction.
Transport of galactose, glucose and their molecular analogues by Escherichia coli K12
Authors: Henderson P J F , Giddens R A , Jones-Mortimer M C
Published: August 2015

1. Strains of Escherichia coli K12 were made that are unable to assimilate glucose by the phosphotransferase system, since they lack the glucose-specific components specified by the genes ptsG and ptsM. 2. Derivative organisms lacking the methyl galactoside or galactose-specific transport system were examined for their ability to transport galactose, d-fucose, methyl beta-D-galactoside, glucose, 2-deoxy-D-glucose and methyl alpha-D-glucoside. 3. Galactose, glucose and to a lesser extent fucose are substrates for both transport systems. 4. 2-Deoxyglucose is transported on the galactose-specific but not the methyl galactoside system. 5. The ability of sugars to elicit anaerobic proton transport is associated with the galactose-specific, but not with the methyl galactoside transport activity. Hence a chemiosmotic mechanism of energization is likely to apply to the former but not to the latter. Alternatively the methyl galactoside system may be switched off under certain conditions, which would indicate a novel regulatory mechanism. 6. Details of the procedure for the derivation of strains may be obtained from the authors, and have been deposited as Supplementary Publication SUP 50074 (8 pages at the) British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977), 161,1.
Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid
Authors: Heckels J E , Lambert P A , Baddiley J
Published: August 2015

When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.
Changes in protein kinase activity in rat testes during development
Authors: Bernard E A , Wassermann G F
Published: August 2015

Protein kinase activity in rat testes remained fairly constant from day 16 1/2 of embryonic life up to 10 days after birth. At the 21st postnatal day a nadir of activity was observed, and after an increase at 35 days of age a decrease in activity at 60 days was seen. The enzyme reached maximal specific activity in the testes of 90-day-old rats.
Properties of glutathione release observed during reduction of organic hydroperoxide, demethylation of aminopyrine and oxidation of some substances in perfused rat liver, and their implications for the physiological function of catalase
Authors: Oshino N , Chance B
Published: August 2015

The enhanced reduction of t-butyl hydroperoxide by glutathione peroxidase is accompanied by a decrease in the cellular concentration of both glutathione and NADPH in isolated liver cells, resulting in the release of GSSG (oxidized glutathione) from the perfused rat liver. This phenomenon, first reported by H. Sies, C. Gerstenecker, H. Menzel & L. Flohé (1972) (FEBS Lett. 27, 171-175), can be observed under a variety of conditions, not only with the acceleration of the glutathione peroxidase reaction by organic peroxides, but also during the oxidation of glycollate and benzylamine, during demethylation of aminopyrine in the liver of the phenobarbital-pretreated rat and during oxidation of uric acid in the liver of the starved rat pretreated with 3-amino-1,2,4-triazole. The rate of release of GSSG is altered markedly by changes in the metabolic conditions which affect the rate of hepatic NADPH generation. Thus, regardless of whether achieved by enhanced oxidation of glutathione by glutathione peroxidase or by oxidation of NADPH through other metabolic pathways, an increase in the cellular concentration of GSSG appears to facilitate its release. It has been found that, in addition to the hexose monophosphate shunt, the mitochondrial NADH-NADP+ transhydrogenase reaction plays an important role in supplying reducing equivalents to the glutathione peroxidase reaction and in maintaining the cellular oxidation-reduction state of the nicotinamide nucleotides. Spectrophotometric analysis of the steady-state concentration of the catalase-H2O2 intermediate with simultaneous measurement of the rate of release of GSSG leads to the conclusion that intracellular compartmentation of catalase in the peroxisomes and glutathione peroxidase in the cytosol and mitochondria distinguishes the reactivities of these enzymes one from the other, and facilitates their effective cooperation in hydroperoxide metabolism in the liver.
Metabolism of glucose, fructose and lactate in vivo in chronically cannulated foetuses and in suckling lambs
Authors: Warnes D M , Seamark R F , Ballard F J
Published: August 2015

1. Chronically cannulated sheep foetuses and suckling lambs were injected with 14C-labelled glucose, fructose or lactate, and sequential blood samples taken under conditions of minimal stress and without anaesthesia. 2. Gluconeogenesis from lactate was not detectable in foetal sheep, but the pathway was active in suckling lambs. 3. Fructose utilization rates were low in foetal sheep, with no measurable conversion into glucose or lactate. 4. The high rates of irreversible loss of both glucose and lactate in the foetus were decreased in suckling lambs. Radioactivity from labelled glucose entered both the lactate and fructose pools in foetal sheep, and entered the lactate pool in suckling lambs. 5. A model is proposed in which carbon flow between glucose, fructose and lactate has been quantified in foetal sheep.
Changes in mammary-gland acetyl-coenzyme A carboxylase associated with lactogenic differentiation
Authors: Mackall Julia C. , Lane M. Daniel
Published: August 2015

The process leading to the rise of acetyl-CoA carboxylase activity in rat mammary tissue after the onset of lactation was investigated. The kinetics of change in enzyme activity and enzyme immunotitratable with antibody against avian liver acetyl-CoA carboxylase were determined during the course of lactogenic differentiation. The antibody inactivates and specifically precipitates acetyl-CoA carboxylase from rat mammary tissue as well as that from chicken liver cytosol. Characterization of the immunoprecipitate of the mammary tissue carboxylase by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals a single biotin-containing polypeptide of about 230000mol.wt. This molecular weight is approximately twice that reported for the avian liver enzyme. However, chicken liver cytosol prepared in the presence of trypsin inhibitor and subjected to immunoprecipitation gives rise to a biotin-containing subunit of 230000mol.wt. as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; omission of proteinase inhibitor leads to a subunit(s) approximately one-half this size. Throughout gestation both carboxylase activity and amounts of immunotitratable enzyme remained low; however, after parturition both parameters rose concomitantly to values 30–40 times the initial values. Therefore the elevated concentration of acetyl-CoA carboxylase appears to result from an increased rate of synthesis of enzyme relative to degradation rather than to activation of a pre-existing form of the enzyme.
The apparent absence of involvement of biotin in the vitamin K-dependent carboxylation of glutamic acid residues of proteins
Authors: Friedman P A , Shia M A
Published: August 2015

The mechanism of the vitamin K-dependent post-translational carboxylation of the gamma-carbon atom of glutamic acid residues in proteins remains obscure. Experiments were performed in vivo and in vitro in an attempt to establish a role for biotin in the transfer of the carboxyl group. Weanling male rats were fed on a biotin-deficient diet until severe biotin deficiency was induced. Their degree of biotin deficiency was documented by assaying for liver acetyl-CoA carboxylase activity, which was about 15% of normal. However, one-stage and two-stage prothrombin times measured on the plasmas were normal. In addition, the liver microsomal fraction did not contain any more prothrombin precursor than did that of normal rat liver. Experiments were done in vitro in which vitamin K-dependent fixing of 14CO2 was measured in the liver microsomal fraction from vitamin K-deficient male rats in the presence or absence of avidin. No evidence for an avidin-sensitive critical biotin-containing site was obtained. Thus neither series of experiments suggests a role for biotin; the data are compatible with carboxyl transfer occurring either through a carboxylated vitamin K intermediate; or via a yet to be identified intermediate, or perhaps via CO2 itself.
Evolution of enzyme catalytic power. Characteristics of optimal catalysis evaluated for the simplest plausible kinetic model
Authors: Brocklehurst Keith
Published: August 2015

1. Evolutionary changes in the structure of an enzyme that provide an increase in its Km value are considered. Provided that Km increases as a result of increases in the forward rate constants of the catalysis relative to the reverse rate constants, the enzyme catalyses the conversion of a fixed concentration of its substrate more rapidly when its structure provides that Km>[S] than when Km<[S]. 2. Catalytic efficiency of enzymes is discussed in terms of the simplest plausible model, the Haldane [(1930) Enzymes, Longmans, London] reversible three-step model: [Formula: see text] The rate equation for the forward reaction of this model (formation of P) may be written in the simple form: [Formula: see text] Keq. is the equilibrium constant (=[P]eq./[S]eq.), and kcat.=V/[E]T, where [E]T is the total enzyme concentration. 3. To assess the effectiveness of an enzyme, it is necessary only to determine the extent to which the constraints of a particular kinetic mechanism permit v2 (v when Km»[S]) to approach vd (the diffusion-limited rate). 4. The value of the optimal rate of catalysis (vopt., the maximal value of v2) is dictated by the equilibrium constant for the reaction, Keq.; v2=vd/a, where [Formula: see text] when k+1 is assumed equal to k−3, and vopt.=vd/amin.. When Keq.≥1, it is necessary that k+2»k−1 for a to take its minimum value, amin.; when Keq.«1, it is necessary only that k+2»Keq.·k−1, i.e. a can equal amin. even if k+2<k−1. When Keq.»1, vopt.=vd; when Keq.=1, vopt.=vd/2, and when Keq.«1, vopt.=Keq.·vd. 5. The analysis, together with predicted effects of evolutionary pressure, suggests that in practice the rates of the fastest enzyme-catalysed freely reversible reactions might be expected to be lower than the value of k+1[E]T[S] by about an order of magnitude, particularly if Keq.<1. 6. The existing literature suggests that, in general, appropriate values of Km have evolved for the provision of high rates of catalysis but that many values of kcat. are not large enough to provide optimal rates of catalysis unless the value of k+1in vivo is lower than its value in free solution.
The structure and enzymic activities of the C1r and C1s subcomponents of C1, the first component of human serum complement
Authors: Sim R B , Porter R R , Reid K B M , Gigli I
Published: August 2015

The subcomponents C1r and C1s and their activated forms C-1r and C-1s were each found to have mol.wts. in dissociating solvents of about 83000. The amino acid compositions of each were similar, but there were significant differences in the monosaccharide analyses of subcomponents C1r and C1s, whether activated or not. Subcomponents C1r and C1s have only one polypeptide chain, but subcomponents C-1r and C-1s each contain two peptide chains of approx. mol.wts. 56000 (“a” chain) and 27000 (“b” chain). The amino acid analyses of the “a” chains from each activated subcomponent are similar, as are those of the “b” chains. The N-terminal amino acid sequence of 29 residues of the C-1s “a” chain was determined, but the C-1r “a” chain has blocked N-terminal amino acid. The 20 N-terminal residues of both “b” chains are similar, but not identical, and both show obvious homology with other serine proteinases. The difference in polysaccharide content of the subcomponents C-1r and C-1s is most marked in the ‘b’ chains. When tested on synthetic amino acid esters, subcomponent C-1r hydrolysed both lysine and tyrosine ester bonds, but subcomponent C-1r did not hydrolyse any amino acid esters tested nor any protein substrate except subcomponent C1s. The lysine esterase activity of subcomponent C1s provides a rapid and sensitive assay of the subcomponent.
A serological investigation into the acidic α-d-mannosidase in normal Angus cattle and in a calf with mannosidosis
Authors: Phillips N C , Winchester B G , Jolly R D
Published: August 2015

Antiserum was raised in a rabbit against bovine kidney acidic alpha-mannosidase that had been purified 570-fold by affinity chromatography on concanavalin A--Sepharose and Sepharose 4B-xi-aminohexanoylmannosylamine. The antiserum precipitated the acidic but not the neutral alpha-mannosidase in normal calf tissues. Human acidic alpha-mannosidase cross-reacted partially with the antiserum, emphasizing the close structural resemblance between the enzyme in the two species. The residual acidic alpha-mannosidase in the tissues of a calf with mannosidosis was also precipitated by the antiserum, the same volume of antiserum being required to precipitate a unit of alpha-mannosidase activity from the normal and pathological tissues. The concentration of cross-reacting material detected by antibody-consumption experiments in the organs of the calf with mannosidosis appeared to be proportional to the concentration of the residual acidic alpha-mannosidase. It is suggested that the residual acidic alpha-mannosidase in mannosidosis accounts for the cross-reacting material detected and that it is unlikely that enzymically inactive but cross-reacting material is present. The residual acidic alpha-mannosidase could be a decreased concentration of the normal gene product or an altered enzyme with a decreased specific enzymic activity and a correspondingly decreased antigenicity.
The action of proteolytic enzymes on the glycoprotein from pig gastric mucus
Authors: Scawen M , Allen A
Published: August 2015

A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.
Multiple α-mannosidase activities in mammalian tissues as shown by metal-ion activation
Authors: Snaith S M
Published: August 2015

1. Four different types of alpha-mannosidase activity were shown to occur in several tissues from the rat. There is the Zn2+-dependent enzyme, active at acidic pH, and three enzymes that are active near to neutral pH. 2. The ‘neutral’ enzymes are activated by Fe2+, Co2+ or Mn2+. 3. Optimum activities for these three enzymes are shown at pH values of 5.2, 6.5 and 7.3. The activity at pH6.5 is the only one evident without metal-ion activation, but activity is enhanced by all three metal ions. The activity at pH 5.2 is seen only in the presence of Fe2+ or Co2+, and the activity at pH7.3 is seen only in the presence of Co2+ or Mn2+ and in a non-chelating buffer medium. 4. The pH6.5-active enzyme is inactivated by EDTA, but activity is restored by excess of metal ion. 5. The enzymes differ markedly in their stability. The pH6.5-active enzyme is very labile and the pH7.3-active enzyme is the most stable. 6. Tissue preparations vary widely in their activity at pH6.5, but where activity is low it can be increased by incubation with one of the activating metal cations. 7. All the enzymes active at neutral pH are inhibited by heavy-metal ions and stabilized to some extent by thiol groups.
A rapid procedure for selectively isolating the major glycoprotein from purified rat brain myelin
Authors: Quarles R H , Pasnak C F
Published: August 2015

Treatment of the chloroform/methanol-insoluble residue of rat brain myelin with lithium 3,5-di-iodosalicylate solubilized the major myelin-associated glycoprotein along with most other proteins and glycoproteins. Equilibration of the extract with phenol resulted in the selective partitioning of the major glycoprotein into the aqueous phase, whereas nearly all of the other proteins and glycoproteins went into the phenol phase.
Steady-state metabolism and transport of d-glucose by rat small intestine in vitro
Authors: Pritchard Penelope J. , Porteous John W.
Published: August 2015

1. Conditions of incubation of everted sacs of rat small intestine were selected to ensure that absorption of d-glucose by mucosal tissue from the incubation medium, intracellular metabolism of the absorbed glucose and transport of glucose through the intact intestinal tissue proceeded linearly with respect to time of incubation within stated time intervals. 2. Under these experimental conditions, steady intracellular concentrations of glucose and lactate were demonstrated. 3. The quantitative translocational and metabolic fate of absorbed glucose was determined under these steady-state conditions. About 25% of glucose absorbed from the external mucosal solution was accumulated (temporarily) within mucosal tissue and about 25% transported through the intact tissue into the external serosal solution; the remainder (about 50%) of the absorbed glucose was metabolized, 90% to lactate and 10% to CO2. Concomitant respiration rates were comparable with those reported for several other preparations of intestine and were stoicheiometrically in excess of the O2 metabolism required to account for the production of CO2 from the absorbed glucose. 4. Water transport through the everted sacs proceeded at an optimum rate under the experimental conditions selected. 5. Some other observations are recorded which influenced the design of the experiments and the interpretation of results; these include the initial physiological state of the animal, the anaesthetic used and the ionic composition of the incubation medium.
Proton translocation coupled to electron flow from endogenous substrates to fumarate in anaerobically grown Escherichia coli K12
Authors: Gutowski S J , Rosenberg H
Published: August 2015

Observed leads to H+/2e- values for proton translocation during the reduction of fumarate by endogenous substrates in anaerobic cells of Escherichia coli K12 varied with fumarate concentration. This variation was probably due mainly to incomplete fumarate utilization. Under optimum conditions a minimum value for leads to H+/2e- of 1.04+/-0.20 was obtained.
Role of haem in the synthesis and assembly of cytochrome P-450
Authors: Bhat Kolari S. , Sardana Mohinder K. , Padmanaban Govindarajan
Published: August 2015

By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide, a drug that degrades the haem moiety of cytochrome P-450, the involvement of haem in cytochrome P-450 synthesis and assembly was investigated. Phenobarbital was used to stimulate apo-(cytochrome P-450) synthesis. Degradation of preformed cytochrome P-450 haem does not result in a concomitant release of the apoprotein from the endoplasmic reticulum. The availability of haem for cytochrome P-450 synthesis in the normal animal is not rate-limiting. Prolonged inhibition of haem synthesis in vivo decreases the rate of apo-(cytochrome P-450) synthesis, although this effect is not discernible under conditions of short-term inhibition of haem synthesis. Under the former conditions exogenous haemin is able to counteract the decrease in the rate of apoprotein synthesis. In animals receiving successive injections of phenobarbital plus 3-amino-1,2,4-triazole, compared with those receiving phenobarbital only, the holo-(cytochrome P-450) content measured spectrally shows a greater decrease than could be accounted for by the decrease in the content of the total apoprotein. In addition to less haem being available under these conditions, the free apoprotein appears to have undergone some modification, such that its haem-binding capacity is considerably decreased. This particular effect could be due to a direct interaction of 3-amino-1,2,4-triazole or its metabolites with cytochrome P-450 rather than a consequence of haem deficiency. Apo-(cytochrome P-450) is capable of binding to the endoplasmic reticulum in a form and at a site, which can be reconstituted with haemin to yield the functional protein.
The stimulus-secretion coupling of glucose-induced insulin release. Insulin release due to glycogenolysis in glucose-deprived islets
Authors: Malaisse W J , Sener A , Koser M , Ravazzola M , Malaisse-Lagae F
Published: August 2015

1. When pancreatic islets are preincubated for 20h in the presence of glucose (83.3mM) and thereafter transferred to a glucose-free medium, theophylline (1.4mM) provokes a dramatic stimulation of insulin release. This phenomenon does not occur when the islets are preincubated for either 20h at low glucose concentration (5.6mM) or only 30 min at the high glucose concentration (83.3mM). 2. The insulinotropic action of theophylline cannot be attributed to contamination of the islets with exogenous glucose and is not suppressed by mannoheptulose. 3. The secretory response to theophylline is an immediate phenomenon, but disappears after 60min of exposure to the drug. 4. The release of insulin evoked by theophylline is abolished in calcium-depleted media containing EGTA. Theophylline enhances the net uptake of 45Ca by the islets. 5. Glycogen accumulates in the islets during the preincubation period, as judged by both ultrastructural and biochemical criteria. Theophylline significantly increases the rate of glycogenolysis during the final incubation in the glucose-free medium. 6. The theophylline-induced increase in glycogenolysis coincides with a higher rate of both lactate output and oxidation of endogenous 14C-labelled substrates. 7. These data suggest that stimulation of glycolysis from endogenous stores of glycogen is sufficient to provoke insulin release even in glucose-deprived islets, as if the binding of extracellular glucose to hypothetical plasma-membrane glucoreceptors is not an essential feature of the stimulus-secretion coupling process.
The metabolism of cyclohexanecarboxylate in the rat
Authors: Brewster D , Jones R S , Parke D V
Published: August 2015

In the rat, cyclohexanecarboxylate was metabolized and excreted (mostly in the urine) as hippurate, hexahydrohippurate, 3,4,5,6-tetrahydrohippurate and benzoyl and cyclohexylcarbonyl beta-glucuronides. The pattern of metabolism is dose-dependent. With decreasing dose a progressive increase in the conversion into hippurate occurred. This was largely at the expense of glucuronide formation, although the proportions of hexahydro- and tetrahydro-hippurate were also decreased. The observed formation of hexahydrohippurate and 3,4,5,5-tetrahydrohippurate substantiates the proposed mechanism of aromatization of cyclohexanecarboxylate. It appears that these compounds arise via glycine conjugation of active intermediates in the aromatization process. Hexahydrohippurate and 3,4,5,6-tetrahydrohippurate may occur in the urine of rats as new mitabolities of shikimate, dependent for their formation on microbial metabolism.
Effects of 5-bromo-2'-deoxyuridine on beating heart cell cultures from neonatal hamsters
Authors: Coetzee G A , Van der Westhuyzen D R , Gevers W
Published: August 2015

1. Primary heart cell cultures from neonatal hamsters yielded a heterogeneous cell population, containing muscle cells undergoing progressive differentiation, as well as non-muscle cells. 2. Addition of 5-bromo-2'-deoxyuridine, at an early stage, to such cultures enhanced the formation of beating sheets of differentiated muscle cells. Accumulation of myosin heavy chains and creatine kinase also occurred in the presence of the analogue. 3. To obtain these effects, the analogue had to be added during the initial rapid growth phase of the cells. Division of the treated cells then ceased when the cell numbers had approximately doubled. 4. Similar results were obtained with other inhibitors of DNA synthesis. Thus improved muscle cell cultures can be obtained by preventing non-muscle cells from overgrowing the cultures. 5. One effect caused only by 5-bromo-2'-deoxyuridine was a large increase in the Ca2+-stimulated ATPase (adenosine triphosphatase) activity which sedimented at low ionic strength. This increase was not due to a greater content of myofibrillar myosin, or to myosin isoenzyme changes, because purified myosin prepared from treated and untreated cultures did not exhibit the increased Ca2+-stimulated ATPase activity.
Pyruvate dehydrogenase activity in hamster small intestine during development
Authors: Schiller C M
Published: August 2015

Total pyruvate dehydrogenase activities in hamster intestine increase from 40 nmol/min (munits) per g of intestine in the foetal animals to 460 munits/g in the adult, whereas the fraction of the enzyme in the active form increases from 34 to 42% of the total activity over the same period. However, a complete conversion of the enzyme into the active form is observed in the neonatal animal immediately after birth. Results from experiments in vitro suggested that the activation of pyruvate dehydrogenase is controlled, in part, by the [NAD+]/[NADH] ratio. This proposal was tested in vivo by examining the proportion of the enzyme in the active form during conditions when the [NAD+]/[NADH] ratio was markedly altered, and the data show a direct relationship between the mitochondrial redox state and activity of the active form.
Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue β-93
Authors: Winterbourn C C , Carrell R W
Published: August 2015

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.
Restoration of β-galactosidase to Escherichia coli M15. Complementation studies
Authors: Marinkovic Dobrivoje V. , Marinkovic Jelka N.
Published: August 2015

Carboxymethylated β-galactosidase from Escherichia coli was dissociated at 100°C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore β-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ‘complementable fractions’. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the β-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented β-galactosidase can exist.
Isolation and characterization of membrane receptors for pokeweed mitogens from mouse lymphocytes
Authors: Yokoyama K , Terao T , Osawa T
Published: August 2015

The major glycoproteins that bind pokeweek B-cell mitogen (Pa-1) and pokeweed T-cell mitogen (Pa-2) were isolated and identified from bone-marrow-derived lymphocytes (B-cells) and thymus-derived lymphocytes (T-cells) of C3H/He mice. The surfaces of the cells were 125I-labelled by using the enzyme lactoperoxidase, and the plasma membranes were isolated from the 125I-labelled cells. These membranes were solubilized with Triton X-100 and subjected to affinity chromatography on the affinity adsorbent prepared by coupling mitogen Pa-1 or Pa-2 to activated Sepharose 4B. The glycoproteins specifically eluted with di-N-acetylchitobiose from the affinity adsorbents were analysed according to their mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate. These glycoproteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes, but they were not detected in the eluate from the T-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were found to be major receptor sites for the pokeweed mitogens on both B-cells and T-cells. However, mitogen Pa-1 (B-cell) has a stronger affinity to Ia antigens than does mitogen Pa-2 (T-cell).
Characterization of glyoxalase I purified from pig erythrocytes by affinity chromatography
Authors: Aronsson Anne-Charlotte , Mannervik Bengt
Published: August 2015

Glyoxalase I (EC 4.4.1.5) was purified about 10000-fold from pig erythrocytes in a yield of approx. 20%. The purification included affinity chromatography on S-hexylglutathione coupled to Sepharose 4B. The purified enzyme normally contained two catalytically active components which were resolved by polyacrylamide-gel electrophoresis. After treatment with reduced glutathione only one component was found. The two components were also demonstrable after isoelectric focusing or DEAE-cellulose chromatography and could also in these cases be fused into one species by preincubation with reduced glutathione. It is proposed that the most acidic form of glyoxalase I is a mixed disulphide with glutathione. Except for these interconvertible forms, the purified enzyme was homogeneous, as judged by disc electrophoresis and sodium dodecyl sulphate/polyacrylamidegel electrophoresis. The molecule is a dimer (48000 daltons), composed of apparently identical subunits (24000 daltons). The isoelectric point was 4.8 at 4°C. The amino acid composition was consistent with the low isoelectric point. The enzyme contained about two thiol groups per enzyme molecule. EDTA inactivated the enzyme and bivalent metal ions could restore fully or partially the catalytic activity; Mg2+ and Mn2+ gave highest activity. It is proposed that a major biological function of glyoxalase I is the detoxification of methylglyoxal formed by enterobacteria in the alimentary canal.