bluuebunny/crossref_metadata_2025 huggingface dataset:
Light-scattering studies on deoxyribonucleic acid flexibility. The solution properties of a small circular deoxyribonucleic acid molecule
Authors: Jolly Douglas J. , Campbell Ailsa M.
Published: August 2015

Previous investigations on the persistence length of DNA in solution have revealed large discrepancies between hydrodynamic results and those from light-scattering techniques which have potentially a greater resolving power. The information obtained from experiments on a small circular DNA molecule has resolved these discrepancies. The non-superhelical circular double-stranded DNA molecule from bacteriophage [unk]X174-infected cells is small enough to permit accurate light-scattering extrapolations, and its solutions have negligible anisotropy. The persistence length obtained from experimental investigations on this molecule is comparable with that obtained by hydrodynamic techniques, even with variation of the excluded-volume factor.
A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates
Authors: Dickinson F. M. , Monger G. P.
Published: August 2015

1. The kinetics of oxidation of ethanol, propan-1-ol, butan-1-ol and propan-2-ol by NAD+ and of reduction of acetaldehyde and butyraldehyde by NADH catalysed by yeast alcohol dehydrogenase were studied. 2. Results for the aldehyde–NADH reactions are consistent with a compulsory-order mechanism with the rate-limiting step being the dissociation of the product enzyme–NAD+ complex. In contrast the results for the alcohol–NAD+ reactions indicate that some dissociation of coenzyme from the active enzyme–NAD+–alcohol ternary complexes must occur and that the mechanism is not strictly compulsory-order. The rate-limiting step in ethanol oxidation is the dissociation of the product enzyme–NADH complex but with the other alcohols it is probably the catalytic interconversion of ternary complexes. 3. The rate constants describing the combination of NAD+ and NADH with the enzyme and the dissociations of these coenzymes from binary complexes with the enzyme were measured.
Studies on the interaction between rabbit liver pyruvate kinase and its allosteric effector fructose 1,6-diphosphate
Authors: Irving M. G. , Williams J. F.
Published: August 2015

Preparation of the L form of rabbit liver pyruvate kinase (EC 2.7.1.40) in the presence of fructose 1,6-diphosphate yielded an enzyme which was kinetically identical with the M or muscle-type form of pyruvate kinase found in liver. Chromatographic and dialysis studies of this complex showed that most of the fructose 1,6-diphosphate molecules were loosely bound to the enzyme, but dilution–dissociation studies and binding experiments established that there was a high initial affinity between the enzyme and fructose 1,6-diphosphate (Kassoc.+2.3×109), and that binding of the loosely bound fructose 1,6-diphosphate was concentration-dependent and a necessary condition to overcome the co-operative interaction observed with the homotropic effector phosphoenolpyruvate. Preparation of the liver enzyme in the absence of EDTA did not yield a predominantly M form of the enzyme, and incubation of the M form in the presence of EDTA did not convert it into the L form, but resulted in inhibition of enzyme activity. Immunological studies confirmed that the L and M forms in liver were distinct, and that preparation of the L form in the presence of fructose 1,6-diphosphate did not produce an enzyme antigenically different from the L form prepared in the absence of this heterotropic effector.
The modification of cholinesterase activity by 5, 5′-dithiobis-(2-nitrobenzoic acid) included in the coupled spectrophotometric assay. Evidence for a non-catalytic substrate-binding site
Authors: Brownson C. , Watts D. C.
Published: August 2015

1. Compared with the acetylcholinesterase assay carried out in the absence of a dithiol, the presence of 5,5′-dithiobis-(2-nitrobenzoic acid) caused marked activation, 6,6′-dithiodinicotinic acid and 2,2′-dithiobis-(5-nitropyridine) less so and 2,2′-dithiodipyridine (aldrithiol-2) had no effect at all. Measurements are further complicated in that the 5-thio-2-nitrobenzoate ion also appears to interact with the enzyme, resulting in slightly lowered absorbance values. 2. Acetylthiocholine competes for the 5,5′-dithiobis-(2-nitrobenzoic acid)-binding site so that activation is essentially eliminated by saturating concentrations of substrate. The presence of the dithiol decreases the Km value of acetylthiocholine. 3. Similar results were obtained with pseudocholinesterase. However, with butyrylthiocholine clear activation was still observed under Vmax. conditions in addition to Km being lowered. 4. All the data yielded Hill coefficients of 1 and analysis of the results leads to the conclusion that activation results from the dithiol being bound to a site on the subunit that is actively catalysing ester hydrolysis. 5. The use of aldrithiol-2 is recommended for kinetic work where absolute quantitative measurements are required.
Regulation of phosphoenolpyruvate carboxylase of Zea mays by metabolites
Authors: Wong K. F. , Davies D. D.
Published: August 2015

Crude preparations of phosphoenolpyruvate carboxylase obtained from aetiolated seedlings of Zea mays are unstable but can be stabilized with glycerol. At the pH optimum of 8.3, the Km value for phosphoenolpyruvate is 80μm. When assayed at 30°C, the enzyme shows normal Michaelis–Menten kinetics, but when assayed at 45°C sigmoid kinetics are exhibited. At pH7.0 the enzyme is inhibited by a number of dicarboxylic acids and by glutamate and aspartate. d and l forms of the hydroxy acids and amino acids are inhibitory and the kinetics approximate to simple non-competitive inhibition. The same compounds produce less inhibition at pH7.6 than at pH7.0 and the kinetics of inhibition are more complex. The enzyme is activated by Pi, by SO42- and by a number of sugar phosphates. Maximum activation occurs at acid pH values, where enzyme activity is lowest. The enzyme is activated by AMP and inhibited by ADP and ATP so that the response to energy charge is of the R type and is thus at variance with Atkinson's (1968) concept of energy charge. The physiological significance of the response to metabolites is discussed.
Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs
Authors: Grewaal D. S. , Quastel J. H.
Published: August 2015

1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke–bicarbonate–[U-14C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K+-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na+ and Ca2+ in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K+, ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na+). 2. Stimulation by K+ of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na+, but some synthesis and release of radioactive acetylcholine occurred in the absence of Na+ as well as in the absence of Ca2+. 3. The Na+ dependence of K+-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na+ lack, and partly due to the suppression of the activity of the Na+ pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca2+ from the incubation medium. It completely blocked K+ stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K+, sodium l-glutamate and lack of Ca2+ on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took place. In the presence of 4mm-KCl it caused some increase in the release of labelled acetylcholine. 7. The barbiturates (Amytal, pentothal), whilst having no significant effects on labelled acetylcholine synthesis in unstimulated brain except at high concentration (1mm), diminished or abolished (at 0.25 or 0.5mm) the enhanced release of acetylcholine, due to high K+ or lack of Ca2+. The fall in tissue content of acetylcholine, due to lack of Ca2+, was diminished or abolished by pentothal (0.25 or 0.5mm) or Amytal (0.25mm).
Transport of glutamine and glutamate in kidney mitochondria in relation to glutamine deamidation
Authors: Crompton M. , Chappell J. B.
Published: August 2015

1. In the absence of added ADP glutamine is transformed by pig kidney mitochondria to ammonium glutamate, which appears in the external medium. This reaction is stimulated only slightly by the addition of ADP, but under these conditions about 20% of the glutamate is oxidized to aspartate. 2. Externally added glutamate is oxidized to aspartate, and at about the same rate as glutamine. 3. The net rates of glutamine and glutamate influx into the intramitochondrial compartment are very slow. 4. The phosphate-dependent glutaminase activity of intact mitochondria is stimulated by the provision of energy. 5. The provision of energy also decreases the concentration of glutamate and increases the concentration of glutamine in the intramitochondrial compartment. These energy-linked changes in the glutamine and glutamate concentrations are of equal magnitude. 6. It is suggested that transport of glutamine and glutamate across the inner membrane of kidney mitochondria occurs by an obligatory exchange between the two metabolites, and is electrogenic. The existence of an electrogenic glutamine–glutamate anti-porter is proposed.
Multiple N-acetyltransferases and drug metabolism. Tissue distribution, characterization and significance of mammalian N-acetyltransferase
Authors: Hearse D. J. , Weber W. W.
Published: August 2015

Investigations in the rabbit have indicated the existence of more than one N-acetyltransferase (EC 2.3.1.5). At least two enzymes, possibly isoenzymes, were partially characterized. The enzymes differed in their tissue distribution, substrate specificity, stability and pH characteristics. One of the enzymes was primarily associated with liver and gut and catalysed the acetylation of a wide range of drugs and foreign compounds, e.g. isoniazid, p-aminobenzoic acid, sulphamethazine and sulphadiazine. The activity of this enzyme corresponded to the well-characterized polymorphic trait of isoniazid acetylation, and determined whether individuals were classified as either `rapid' or `slow' acetylators. Another enzyme activity found in extrahepatic tissues readily catalysed the acetylation of p-aminobenzoic acid but was much less active towards isoniazid and sulphamethazine. The activity of this enzyme remained relatively constant from individual to individual. Studies in vitro and in vivo with both `rapid' and `slow' acetylator rabbits revealed that, for certain substrates, extrahepatic N-acetyltransferase contributes significantly to the total acetylating capacity of the individual. The possible significance and applicability of these findings to drug metabolism and acetylation polymorphism in man is discussed.
The oxoacyl-coenzyme A thiolases of animal tissues
Authors: Middleton B.
Published: August 2015

1. The activities and relative 3-oxoacyl-CoA substrate specificities of oxoacyl-CoA thiolase were determined in a large number of animal tissues. The relative activities with different 3-oxoacyl-CoA substrates varied widely in different tissues and, in addition, the activity as measured with acetoacetyl-CoA (but not with other longer-carbon-chain acyl-CoA substrates) was activated by K+. 2. These properties were due to the presence, in different proportions in each tissue, of three classes of thiolase, all of which use acetoacetyl-CoA as substrate but which have different intracellular locations and substrate specificities and which differ also in kinetic and chromatographic behaviour. 3. Cytoplasmic thiolase activity was found to be widely distributed among different tissues and was due to an acetoacetyl-CoA-specific thiolase. This cytoplasmic activity was found to account for a significant proportion of the total tissue activity towards acetoacetyl-CoA in several tissues, and especially in the brain of newborn rats. 4. Mitochondrial thiolase activity towards acetoacetyl-CoA was due to two different classes of enzyme whose relative amounts varied with the tissue type. An oxoacyl-CoA thiolase of general specificity for the acyl-CoA substrate constituted one class, the other being a specific acetoacetyl-CoA thiolase that differed from its cytoplasmic counterpart in being greatly stimulated by K+. 5. This activation by K+ made it possible to calculate the tissue contents of mitochondrial acetoacetyl-CoA thiolase and mitochondrial oxoacyl-CoA thiolase from measurements of activity with acetoacetyl-CoA in tissue extracts under defined conditions. 6. The properties and the different thiolases and their tissue distribution is discussed with respect to their possible roles in metabolism.
Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris
Authors: Gray J. C. , Kekwick R. G. O.
Published: August 2015

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a Km of 4.26×10−5m for RS-mevalonate and 1.54×10−3m for ATP. The preparation from green leaves had a Km of 4.55×10−5m for RS-mevalonate and 1.75×10−3m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.
The mechanism of inhibition of rat liver tryptophan pyrrolase activity by 4-hydroxypyrazolo[3,4-d]pyrimidine (allopurinol)
Authors: Badawy Abdulla A.-B. , Evans Myrddin
Published: August 2015

1. Allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine) selectively inhibits the apotryptophan pyrrolase activity in homogenates of rat liver in vitro and after intraperitoneal administration. The inhibition is abolished by an excess of haematin. The allopurinol metabolite alloxanthine has no effect on the pyrrolase activity in vitro or after administration. Allopurinol also inhibits the activation of the enzyme in vitro by ascorbate, ethanol plus NAD+, NADH, hypoxanthine or xanthine. It is suggested that these agents cause the conversion of a latent form of the pyrrolase into the apoenzyme, and that xanthine oxidase is not involved in this process. 2. The raised total pyrrolase activity observed after the administration of cortisol, cyclic AMP, tryptophan, salicylate or ethanol is lowered by allopurinol in vitro to the corresponding holoenzyme values. A similar effect is observed when allopurinol is administered shortly before cortisol or cyclic AMP. Pretreatment of rats with allopurinol completely prevents the enhancement of the pyrrolase activities by tryptophan, salicylate or ethanol. 3. It is suggested that allopurinol inhibits rat liver tryptophan pyrrolase activity in vitro and after administration by preventing the conjugation of the apoenzyme with its haem activator. The possible usefulness of combined allopurinol–tryptophan therapy of affective disorders is discussed.
Inosine 5′-monophosphate dehydrogenase of Escherichia coli K12: the nature of the inhibition by guanosine 5′-monophosphate (Short Communication)
Authors: Lambden Paul R. , Drabble William T.
Published: August 2015

When IMP is the variable substrate, IMP dehydrogenase (EC 1.2.1.14) gives non-linear Lineweaver–Burk plots in the presence of GMP. At 0.1mm-GMP the Hill coefficient n=1.15 and at 0.2mm-GMP n=1.34. In the absence of GMP n=1.0. The fact that n exceeds 1.0 in the presence of GMP indicates that the enzyme possesses regulatory (allosteric) properties. The inhibition by GMP is competitive with respect to IMP and non-competitive with respect to NAD+.
Negatively co-operative ligand binding
Authors: Dixon Henry B. F. , Tipton Keith F.
Published: August 2015

Simple systems are considered in which the binding of a ligand at a single site exhibits a doubly sigmoid curve when saturation is plotted against the logarithm of ligand concentration, i.e. where a fraction of the site exhibits one dissociation constant and the rest exhibits another. The condition for this to occur is that the ligand should also combine at another site on the binding molecule with comparable affinity and that the binding at one site should markedly lower affinity at the other. The protonation of simple compounds such as cysteine and 3-hydroxypyridine is taken as an example of this process and the equations derived are also applied to the binding of substrates to enzymes.
A reappraisal of the effects of adenosine 3′:5′-cyclic monophosphate on the function and morphology of the rat prostate gland
Authors: Mangan F. R. , Pegg A. E. , Mainwaring W. I. P.
Published: August 2015

1. A comparison was made of the binding of 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) and cyclic AMP in the rat prostate gland. Distinct binding mechanisms exist for these compounds, and cyclic AMP cannot serve as a competitor for the 5α-dihydrotestosterone-binding sites and vice versa. In contrast with the results obtained with 5α-dihydrotestosterone, very small amounts of cyclic AMP are retained in nuclear chromatin and the overall binding of this cyclic nucleotide is not markedly affected by castration. 2. Androgenic stimulation does not lead to major increases in the adenylate cyclase activities associated with any subcellular fraction of the prostate gland. Accordingly, changes in the concentration of cyclic AMP in the prostate gland after hormonal treatment are likely to be small, but these were not measured directly. 3. When administered to whole animals in vivo, small amounts of non-degraded cyclic AMP are found in the prostate gland but sufficient to promote an activation of certain carbohydrate-metabolizing enzymes in the cell supernatant fraction. The stimulatory effects of cyclic AMP were not evident with cytoplasmic enzymes engaged in polyamine synthesis or nuclear RNA polymerases. These latter enzymes were stimulated solely by the administration of testosterone. 4. By making use of antiandrogens, a distinction can be drawn between the biochemical responses attributable to the binding of 5α-dihydrotestosterone but not of cyclic AMP. Evidence is presented to suggest that the stimulation of RNA polymerase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase is a consequence of the selective binding of 5α-dihydrotestosterone. Only the stimulation of glucose 6-phosphate dehydrogenase can be attributed to cyclic AMP or other metabolites of testosterone. 5. Overall, this study indicates that the formation of cyclic AMP is not a major feature of the androgenic response and affects only a restricted number of biochemical processes. Certainly, cyclic AMP cannot be considered as interchangeable with testosterone and its metabolites in the control of the function of the prostate gland. This difference is additionally emphasized by the failure of cyclic AMP to restore the morphology of the prostate gland in castrated animals; morphological restoration only follows the administration of androgens.
Studies of cytochrome synthesis in rat liver
Authors: Druyan Robert , Jakovcic Smilja , Rabinowitz Murray
Published: August 2015

The incorporation of radioactive amino acids and of δ-amino[2,3-3H2]laevulinate into rat liver cytochromes b5 and c and cytochrome oxidase has been examined with and without protein-synthesis inhibitors. Cycloheximide promptly inhibits labelling of both haem and protein for cytochrome c in parallel fashion. Although incorporation of 14C-labelled amino acid into microsomal cytochrome b5 is also rapidly inhibited, cycloheximide incompletely inhibits haem labelling of cytochrome b5 and cytochrome a+a3, and inhibition occurs only after repeated antibiotic injections. The possibility of apo-protein pools, or of haem exchange, with a rapidly renewed ‘free’ haem pool, is considered. Consistent with this model is the observation of non-enzymic haem exchange in vitro between cytochrome b5 and methaemoglobin. Chloramphenicol, injected intravenously over 5h, results in a 20–40% decrease in incorporation of δ-amino[2,3-3H2]laevulinate into haem a+a3 and haem of cytochromes b5 and c. With the dosage schedule of chloramphenicol studied, amino acid labelling of total liver protein and of cytochrome c was not inhibited. Similarly, ferrochelatase activity was not decreased.
Metabolism of free and esterified cholesterol by Leydig-cell tumour mitochondria
Authors: Moyle William R. , Jungas Robert L. , Greep Roy O.
Published: August 2015

1. Experiments were designed to localize intracellularly the enzymes and sterol substrates required for steroidogenesis in Leydig-cell tumours. Subcellular fractions were prepared by differential centrifugation of tumour homogenates. Both free and esterified cholesterol were associated primarily with the fractions sedimenting at 1400gav. and the lipid layer floating on the surface of the isolation tubes; they were not found in the mitochondria, where the conversion of cholesterol into pregnenolone occurred. 2. Hydrolysis of esterified cholesterol was required before it could be oxidized to pregnenolone. 3. An enzyme capable of hydrolysing cholesterol esters was located external to the mitochondria. 4. Mitochondria were subfractionated by allowing them to swell in 0.02m-phosphate buffer (pH7.2) and separating the inner and outer membranes by sedimentation in sucrose gradients. The outer membrane, identified by its content of monoamine oxidase, contained most of the cholesterol associated with the mitochondria. The inner membrane, identified by its content of succinate dehydrogenase, contained the cholesterol side-chain-cleaving enzyme and very little cholesterol. 5. Accumulation of sterols by the mitochondria was studied by incubating this fraction with labelled free and esterified cholesterol suspended in lipid-free bovine serum albumin. Two phases of cholesterol accumulation were observed. The first phase, requiring 10–15min, was independent of the incubation temperature, and was inhibited by the presence of bovine serum albumin in the incubation medium. The second phase of accumulation was independent of the serum albumin content of the medium but was inhibited by low incubation temperature. 6. Esterified cholesterol was not accumulated by the mitochondria after the initial rapid binding phase. 7. The findings suggest that cholesterol was not rapidly accumulated by the mitochondrial fraction in vitro and that mechanisms may be required to facilitate cholesterol transport into mitochondria in intact tumour cells during the periods in which steroidogenesis is stimulated maximally.
Metabolic alterations produced in the liver by chronic ethanol administration. Increased oxidative capacity
Authors: Videla L. , Bernstein J. , Israel Y.
Published: August 2015

1. Administration of ethanol (14g/day per kg) for 21–26 days to rats increases the ability of the animals to metabolize ethanol, without concomitant changes in the activities of liver alcohol dehydrogenase or catalase. 2. Liver slices from rats chronically treated with ethanol showed a significant increase (40–60%) in the rate of O2 consumption over that of slices from control animals. The effect of uncoupling agents such as dinitrophenol and arsenate was completely lost after chronic treatment with ethanol. 3. Isolated mitochondria prepared from animals chronically treated with ethanol showed no changes in state 3 or state 4 respiration, ADP/O ratio, respiratory control ratio or in the dinitrophenol effect when succinate was used as substrate. With β-hydroxybutyrate as substrate a small but statistically significant decrease was found in the ADP/O ratio but not in the other parameters or in the dinitrophenol effect. Further, no changes in mitochondrial Mg2+-activated adenosine triphosphatase, dinitrophenol-activated adenosine triphosphatase or in the dinitrophenol-activated adenosine triphosphatase/Mg2+-activated adenosine triphosphatase ratio were found as a result of the chronic ethanol treatment. 4. Liver microsomal NADPH oxidase activity, a H2O2-producing system, was increased by 80–100% by chronic ethanol treatment. Oxidation of formate to CO2in vivo was also increased in these animals. The increase in formate metabolism could theoretically be accounted for by an increased production of H2O2 by the NADPH oxidase system plus formate peroxidation by catalase. However, an increased production of H2O2 and oxidation of ethanol by the catalase system could not account for more than 10–20% of the increased ethanol metabolism in the animals chronically treated with ethanol. 5. Results presented indicate that chronic ethanol ingestion results in a faster mitochondrial O2 consumption in situ suggesting a faster NADH reoxidation. Although only a minor change in mitochondrial coupling was observed with isolated mitochondria, the possibility of an uncoupling in the intact cell cannot be completely discarded. Regardless of the mechanism, these changes could lead to an increased metabolism of ethanol and of other endogenous substrates.
Metabolic alterations produced in the liver by chronic ethanol administration. Changes related to energetic parameters of the cell
Authors: Bernstein J. , Videla L. , Israel Y.
Published: August 2015

1. Chronic ethanol administration to rats for 21–27 days increases the rate of O2 consumption as measured in liver slices. The extra respiration can be abolished by inhibition of the active transport of Na+ and K+. Dinitrophenol activates the respiratory rate in the liver of the treated animals only in the presence of ouabain. 2. Active (ouabain-sensitive) transport of 86Rb and (Na++K+)-stimulated adenosine triphosphatase activity were increased in the livers of the ethanol-treated animals. 3. Chronic ethanol administration also led to a decrease in the phosphorylation potential ([ATP]/[ADP][Pi]) in the liver cell owing to a decrease in [ATP] and an increase in [Pi]. 4. It is suggested that an increased sodium pump activity is responsible for the increased oxidative capacity and for the insensitivity to dinitrophenol observed in the livers of ethanol-treated animals.
Studies on β-glucanases. Some properties of a bacterial endo-β-(1→3)-glucanase system
Authors: Manners David J. , Wilson Glynn
Published: August 2015

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley β-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific β-glucanases, an endo-β-(1→3)-glucanase and an endo-β-(1→4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-β-(1→3)-glucanase having a molecular weight of about 16000.
The structure of a β-(1→3)-d-glucan from yeast cell walls
Authors: Manners David J. , Masson Alan J. , Patterson James C.
Published: August 2015

Yeast glucan as normally prepared by various treatments of yeast (Saccharomyces cerevisiae) cell walls to remove mannan and glycogen is still heterogeneous. The major component (about 85%) is a branched β-(1→3)-glucan of high molecular weight (about 240000) containing 3% of β-(1→6)-glucosidic interchain linkages. The minor component is a branched β-(1→6)-glucan. A comparison of our results with those of other workers suggests that different glucan preparations may differ in the degree of heterogeneity and that the major β-(1→3)-glucan component may vary considerably in degree of branching.
The purification and properties of the lectin from potato tubers, a hydroxyproline-containing glycoprotein
Authors: Allen Anthony K. , Neuberger Albert
Published: August 2015

1. Potato lectin has been purified and shown to be a glycoprotein containing about 50% of carbohydrate. Most of the sugar residues (92%) are arabinose; small amounts of galactose, glucose and glucosamine are also present. 2. The most abundant amino acid is hydroxyproline (16% of the residues), 11.5% of the residues are half-cystine and phenylalanine is absent. The lectin also contains about one residue/molecule of a basic amino acid, not usually found in proteins, which has been tentatively identified as ornithine. There is indirect evidence that the components of the glycoprotein are linked through hydroxyproline and arabinose. 3. By gel filtration in 6m-guanidine–HCl on Sepharose 4B, it was found that both the native glycoprotein and its S-carboxymethylated derivative had subunit molecular weights of 46000 (±5000). In a non-denaturing solution, two of these units appear to be associated. 4. The lectin is specifically inhibited in its agglutination reaction by oligosaccharides that contain N-acetylglucosamine. Its specificity is similar to, but not identical with, that of wheat-germ agglutinin.
γδ-Dioxovalerate as a substrate for the glyoxalase enzyme system
Authors: Jerzykowski Tadeusz , Winter Romana , Matuszewski Wojciech
Published: August 2015

1. Crude γδ-dioxovalerate was synthesized from laevulinate by two different methods and was purified by Sephadex chromatography. Some analytical reactions of the compound are described. 2. γδ-Dioxovalerate is a substrate for glyoxalase I and the GSH derivative formed by this enzyme is hydrolysed by glyoxalase II to form d-α-hydroxyglutarate. The Km of glyoxalase I for γδ-dioxovalerate is 1.0×10−3m at pH5.8.3. The u.v.-absorption spectrum of thiol ester, synthesized enzymically from γδ-dioxovalerate and GSH by glyoxalase I, is almost identical with that for S-lactoylglutathione. Some optical properties of this thiol ester were measured. 4. Attempts to show reversibility of the glyoxalase system reactions with d-α-hydroxyglutarate as substrate were unsuccessful. 5. The possible metabolic role of the γδ-dioxovalerate reaction is discussed. It is suggested that one of the metabolic functions of the glyoxalase system may be to provide a mechanism for the entry of this compound into the tricarboxylic acid cycle.
The reaction pathway of membrane-bound rat liver mitochondrial monoamine oxidase
Authors: Houslay Miles D. , Tipton Keith F.
Published: August 2015

1. A preparation of a partly purified mitochondrial outer-membrane fraction suitable for kinetic investigations of monoamine oxidase is described. 2. An apparatus suitable for varying the O2 concentration in a spectrophotometer cuvette is described. 3. The reaction catalysed by the membrane-bound enzyme is shown to proceed by a double-displacement (Ping Pong) mechanism, and a formal mechanism is proposed. 4. KCN, NaN3, benzyl cyanide and 4-cyanophenol are shown to be reversible inhibitors of the enzyme. 5. The non-linear reciprocal plot obtained with impure preparations of benzylamine, which is typical of high substrate inhibition, is shown to be due to aldehyde contamination of the substrate.
Biosynthesis and degradation of saccharopine, an intermediate of lysine metabolism
Authors: Fellows Florence C. I.
Published: August 2015

Lysine–2-oxoglutarate reductase was prepared from ox liver and its characteristics were examined. Its activity was totally inhibited in the presence of NH4Cl. Under conditions that inhibit saccharopine formation, and in the presence of NADP+, ox liver mitochondria were found to catalyse the hydrolysis of saccharopine to lysine and α-oxoglutarate. The enzyme involved was named saccharopine oxidoreductase. It was partially purified and separated from lysine–oxoglutarate reductase. Comparison of the properties of these two enzymes showed that saccharopine degradation was stimulated under conditions that inhibit its formation. The effect of pH, various cofactors and stability during incubation confirm that saccharopine biosynthesis from, and degradation to, lysine are catalysed by two distinct enzymes.
S-Adenosyl-l-methionine–cycloartenol methyltransferase activity in cell-free systems from Trebouxia sp. and Scenedesmus obliquus
Authors: Wojciechowski Zdzilaw A. , Goad L. John , Goodwin Trevor W.
Published: August 2015

1. Homogenates prepared from Trebouxia sp. 213/3 and Scenedesmus obliquus exhibited S-adenosyl-l-methionine–cycloartenol methyltransferase activity. 2. The products of the reaction, with cycloartenol as the substrate, were 24-methylenecycloartanol and cyclolaudenol. 3. Optimal enzyme activity was found in homogenates prepared at pH7.6 and the transmethylase was distributed between the supernatant and microsomal fractions of the Trebouxia homogenate. 4. The relevance of these results is discussed in relation to C28 and C29 sterol production in the algae.
The design of experiments using isotopes for the determination of the rates of disposal of blood-borne substrates in vivo with special reference to glucose, ketone bodies, free fatty acids and proteins
Authors: Heath Dennis F. , Barton Roger N.
Published: August 2015

1. The two well-known methods of estimating rates of irreversible disposal (R) of blood-borne substrates in vivo by isotope experiments involve estimating the specific radioactivity (S) of the substrate in blood either after single intravenous injection of labelled substrate or during its infusion at a constant rate. The value of R is calculated from the S–time curve, usually by assuming: (i) a metabolic steady state with respect to substrate, (ii) the passage of all substrate through the blood, and (iii) the absence of certain types of recycling via blood. 2. In a theoretical investigation we show how experiments can be performed and R calculated from analyses of blood when one or more of the above assumptions is unjustified, by using glucose, ketone bodies, plasma free fatty acids and proteins as examples. In general the methods require single injection procedures, with estimation of the total quantity of label in the substrate in blood and the substrate concentration instead of only S. Such values give estimates of R with standard errors even when only one blood specimen is taken from each of a group of animals, as is convenient when working with small animals or substrates in low concentration, and when the animals are in a non-steady state in which constant infusion procedures are invalid. 3. Similar methods give the fraction of label injected as one compound which passes through another (the isotopic yield). 4. The methods are not always applicable, and cannot be applied to plasma proteins in some pathological conditions. A questionnaire for assessing their applicability is given.
Role of mixed-function amine oxidase in N-hydroxylation of 2-acetamidofluorene by hamster liver microsomal preparations
Authors: Lotlikar Prabhakar D. , Wertman Kathleen , Luha Leida
Published: August 2015

Pretreatment of hamsters with 3-methylcholanthrene (100mg/kg body wt.) 24h before death did not appreciably change the extent of N-oxide formation when hepatic microsomal preparations were incubated with NN-dimethylaniline as substrate. In contrast, the N-hydroxylation of 2-acetamidofluorene was increased severalfold in hepatic microsomal preparations from pretreated animals. Under these conditions there were no appreciable changes in cytochrome P-450 content and NADPH–cytochrome c reductase activity. On the basis of these comparative data, it is suggested that amine oxidase is not involved in N-hydroxylation of 2-acetamidofluorene.
Inhibition of enzymes by metal ion-chelating reagents. The action of copper-chelating reagents on diamine oxidase
Authors: Bardsley William G. , Childs Robert E. , Crabbe M. James C.
Published: August 2015

1. The inhibition of diamine oxidase has been studied by using the following copper-chelating reagents: 1,10-phenanthroline; 2,2′-bipyridyl; 8-hydroxyquinoline (oxine); diethyldithiocarbamate and dithio-oxamide (rubeanic acid). 2. Addition of chelating reagent caused a rapid inhibition of enzyme to a degree dependent solely on the final inhibitor concentration. Addition of substrate gave linear initial rates of reaction showing that under these conditions the inhibition was not being rapidly reversed. 3. The inhibition has been investigated by using new graphical methods and has been found in all cases to involve the chelating agents completely removing two Cu2+ ions from the enzyme. An alternative possibility, involving ligand substitution, was eliminated. 4. A value of K=8.0×10-33m-2 has been found for the enzyme in equilibrium with 2 Cu2+ ions (i.e. β2, the stability constant for diamine oxidase/two Cu2+, is 32.1).
Mannosidosis in Angus cattle. The enzymic defect
Authors: Phillips N. C. , Robinson D. , Winchester B. G. , Jolly R. D.
Published: August 2015

Normal calf α-mannosidase activity exists in at least three forms separable by chromatography on DEAE-cellulose and by starch-gel electrophoresis. Two components, A and B, have optimum activity between pH3.75 and 4.75, but component C has an optimum of pH6.6. Components A and B are virtually absent from the tissues of a calf with mannosidosis and the residual activity is due to component C. The acidic and neutral forms of α-mannosidase differ in their molecular weights and sensitivity to EDTA, Zn2+, Co2+ and Mn2+. An acidic α-mannosidase component (pH optimum 4.0) accounts for most of the activity in normal plasma but it is absent from the plasma of a calf with mannosidosis. Although the acidic α-mannosidase component is probably related to tissue components A and B, it can be distinguished from them by ion-exchange chromatography and gel filtration. The optimum pH of the low residual activity in the plasma from a calf with mannosidosis is pH5.5–5.75. The results support the hypothesis that Angus-cattle mannosidosis is a storage disease caused by a deficiency of lysosomal acidic α-mannosidase activity.
Short Communication. Weighting functions and data truncation in the fitting of multi-exponential functions
Authors: Atkins Gordon L.
Published: August 2015

Artificial data were simulated by using two-exponential functions and normally distributed pseudo-random numbers. The variation corresponded to either constant or relative variance for the data error. These data were used to test (i) three different weighting functions and (ii) the effect of data truncation on the precision of estimating the parameters of two-exponential functions.
Studies on the subunit structure and amino acid sequence of triose phosphate isomerase from chicken breast muscle
Authors: Furth Anna J. , Milman J. D. , Priddle J. D. , Offord R. E.
Published: August 2015

1. Triose phosphate isomerase was prepared by chromatography on DEAE-cellulose of an (NH4)2SO4 fraction of an extract of homogenized chicken breast muscle. The product is homogeneous on gel electrophoresis and is suitable for growing crystals for X-ray work. The specific activity is 10000 units/mg and the value for E0.1%280 is 1.20. 2. Comparison between the sum of the amino acid compositions of the tryptic peptides of the protein and the amino acid composition obtained on total hydrolysis of the protein indicates that the relative subunit mass is about 27000. 3. These data, together with the results of the examination of the amino acid compositions of a number of minor peptides, the number of peptides in the tryptic digest and the complete amino acid sequences of the tryptic peptides (the determination of which is described here), give no indication that the subunits are dissimilar. 4. A tentative amino acid sequence is presented for the protein, in which the ordering of the tryptic peptides is derived by homology with the sequence of the rabbit muscle enzyme (Corran & Waley, 1973). 5. An appendix describes the use that was made of mass spectrometry in the determination of some of the sequences. Mass-spectrometric data have been obtained for 35 residues, that is about 15% of the total sequence of the protein. 6. An extended version of the present paper has been deposited as Supplementary Publication SUP 50025 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.
The mechanism of action of superoxide dismutase from pulse radiolysis and electron paramagnetic resonance. Evidence that only half the active sites function in catalysis
Authors: Fielden E. Martin , Roberts Peter B. , Bray Robert C. , Lowe David J. , Mautner Gillian N. , Rotilio Giuseppe , Calabrese Lilia
Published: August 2015

1. Detailed studies on the mechanism of the enzymic reaction of bovine superoxide dismutase were carried out by using pulse radiolysis and electron paramagnetic resonance (e.p.r.). 2. The second-order rate constant for reaction between superoxide dismutase and the superoxide ion was redetermined as (2.37±0.18)×109m−1·s−1 at 25°C. This reaction governs the turnover, and any first-order steps must have rate constants higher than about 106s−1. Turnover has a low activation energy and is slowed substantially when the viscosity is increased with glycerol, confirming that the reaction rate is near the limit for diffusion control. In water a reversible conformation change to a less active form appears to take place above about 40°C. 3. Pre-steady-state rates of reduction and reoxidation of copper in the enzyme are consistent with these processes being rate-limiting in enzyme turnover. 4. Examination, with the help of computer simulation, of the e.p.r. spectra at 9 and 35GHz of native superoxide dismutase indicated that, apart from 10–20% of impurities, only one species of Cu2+ is distinguishable. Further, the specific activity of our enzyme preparations, measured by pulse radiolysis, is at least as high as that obtained by other workers. 5. Nevertheless, measurement of the proportion of copper present as Cu2+ (determined both optically and by e.p.r. spectroscopy) in the steady states approached from both the oxidized and the reduced forms of the enzyme, indicates (after allowing for the impurities) that only half of the copper atoms participate in turnover. E.p.r. spectroscopy provided no evidence for differences between functioning and non-functioning Cu2+ atoms. 6. It is suggested that the results may be best interpreted in terms of an allosteric type of mechanism, with two initially indistinguishable copper atoms in the enzyme. Reaction of one of these with a superoxide ion then renders the other, at least transiently, unreactive.
Partial amino acid sequence in the N-terminal region of an anti-pneumococcal immunoglobulin heavy chain of allotype a2
Authors: Jaton Jean-Claude , Haimovich Joseph
Published: August 2015

The amino acid sequence of the N-terminal 48 residues of the heavy chain derived from a homogeneous rabbit antibody to type III pneumococci is described. This chain of allotype a2 is compared with other rabbit heavy chains of allotypes a1, a2 and a3. Within the N-terminal 25 positions, two chains which carry the same allotype a2 possess identical amino acid sequences, but differ markedly from heavy chains of allotypes a1 and a3. Sequence variability is observed in residues 26–27 and 30–34, but not in residues 35–48.
Purification of α-mannosidase from jack-bean meal suitable for use as reagent (Short Communication)
Authors: Chu Clement Y. T. , Turner Michael D.
Published: August 2015

A simple method of purification of α-mannosidase from jack-bean meal is described which yields a product free of β-N-acetylglucosaminidase activity.
The isolation and properties of phenylalanine hydroxylase from human liver
Authors: Woo Savio L. C. , Gillam Shirley Su , Woolf Louis I.
Published: August 2015

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The Km values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.
The uptake and metabolism of [32P]pyrophosphate by mouse calvaria in vitro
Authors: Jung André , Bisaz Sylvia , Gebauer Urs , Russell Robert Graham G. , Morgan David Brian , Fleisch Herbert
Published: August 2015

In order to study the uptake and metabolism of PPi by bone, 32PPi was added to the medium surrounding explanted mouse calvaria maintained in organ culture. Most of the PPi was hydrolysed during incubation, but there was a measurable entry of intact PPi into bone. When 32Pi was added to the medium, synthesis of PPi and organic phosphates from Pi was observed in bone. There was no detectable passage of PPi from bone into the medium. These results are discussed in terms of two models of pyrophosphate hydrolysis and exchange. Some quantitative estimates about the fate of PPi in bone were made.
Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle
Authors: Cummins Peter , Perry S. Victor
Published: August 2015

1. On electrophoresis in dissociating conditions the tropomyosins isolated from skeletal muscles of mammalian, avian and amphibian species migrated as two components. These were comparable with the α and β subunits of tropomyosin present in rabbit skeletal muscle. 2. The α and β components of all skeletal-muscle tropomyosins contained 1 and 2 residues of cysteine per 34000g respectively. 3. The ratio of the amounts of α and β subunit present in skeletal muscle tropomyosins was characteristic for the muscle type. Muscle consisting of slow red fibres contained a greater proportion of β-tropomyosin than muscles consisting predominantly of white fast fibres. 4. Mammalian and avian cardiac muscle tropomyosins consisted of α-tropomyosin only. 5. Mammalian and avian smooth-muscle tropomyosins differed both chemically and immunologically from striated-muscle tropomyosins. 6. Antibody raised against rabbit skeletal α-tropomyosin was species non-specific, reacting with all other striated muscle α-tropomyosin subunits tested. 7. Antibody raised against rabbit skeletal β-tropomyosin subunit was species-specific.