bluuebunny/crossref_metadata_2025 huggingface dataset:
The bacterial degradation of flavonoids. Oxidative fission of the A-ring of dihydrogossypetin by a Pseudomonas sp
Authors: Jeffrey A. M. , Jerina D. M. , Self R. , Evans W. C.
Published: August 2015

Cell-free extracts prepared from a Pseudomonas sp., grown on (+)-catechin, oxidized dihydrogossypetin (3′,4′,5,7,8-pentahydroxyflavanonol) by cleaving the A-ring to form oxaloacetic acid from C-5, C-6, C-7 and C-8 together with 5-(3,4-dihydroxyphenyl)-4-hydroxy-3-oxovalero-δ-lactone. The structure of this lactone was confirmed by synthesis of related phenylvalerolactones.
The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in muscles from vertebrates and invertebrates
Authors: Crabtree B. , Higgins S. J. , Newsholme E. A.
Published: August 2015

1. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in crude homogenates of vertebrate and invertebrate muscles are reported. 2. Pyruvate carboxylase activity was present in all insect flight muscles that were investigated: in homogenates of bumble-bee flight muscle the activity was inhibited by ADP and activated by acetyl-CoA, and it was distributed mainly in the mitochondrial fraction. This is the first demonstration of pyruvate carboxylase activity in muscle. However, the activity appears to be restricted to insect flight muscle, since it was not found in other invertebrate or vertebrate muscles. 3. Since the three enzymes were never found together in the same muscle, it is concluded that these enzymes cannot provide a pathway for the synthesis of glycogen from lactate or pyruvate in muscle. Other roles for these enzymes in muscle are suggested. In particular, pyruvate carboxylase may be present in insect flight muscle for the provision of oxaloacetate to support the large increase in activity of the tricarboxylic acid cycle which occurs when an insect takes flight.
The uptake and metabolism of plasma lysophosphatidylcholine in vivo by the brain of squirrel monkeys
Authors: Illingworth D. R. , Portman O. W.
Published: August 2015

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-14C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-3H]choline; 3H:14Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the 3H:14C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-3H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and βine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.
The location of N-acetylgalactosamine in the walls of Bacillus subtilis 168
Authors: Duckworth M. , Archibald A. R. , Baddiley J.
Published: August 2015

The N-acetylgalactosamine in the walls of Bacillus subtilis 168 occurs in two polymers. One of these contains N-acetylgalactosamine, glucose and phosphorus and is attached to the peptidoglycan through an alkali-labile bond; preliminary studies indicate that a repeating unit of this polymer is glucosyl-N-acetylgalactosamine 1-phosphate. N-Acetylgalactosamine is also associated with the peptidoglycan in a component that is not converted into the free sugar or other soluble compounds on treatment of the walls with alkali. The two polymers containing N-acetylgalactosamine are released on autolysis of the walls and can be separated by ion-exchange chromatography. As glucose 6-phosphate is produced by gentle hydrolysis of the wall with acid a third phosphate polymer, poly(glucose 1-phosphate), may occur in this wall. However, as no polymer with this structure could be separated from that containing galactosamine, its existence has not been established unequivocally. The methods described permit the study of variations in N-acetylgalactosamine content with respect to growth conditions.
Isolation and characterization of membranes from normal and transformed tissue-culture cells
Authors: Graham John M.
Published: August 2015

Homogenates of baby-hamster kidney cells and rat embryo fibroblasts prepared by nitrogen cavitation contain a small population of slowly sedimenting mitochondria or mitochondrial fragments, which contaminate the microsomal fraction. This appears to limit the resolution of surface membrane and endoplasmic reticulum on magnesium-containing dextran gradients. The microsomal material and mitochondria can, however, be completely separated on a 10–60% (w/w) sucrose zonal gradient containing a 30% sucrose plateau. On magnesium-containing dextran gradients this mitochondria-free microsomal material can be resolved into at least two surface membrane fractions and at least two endoplasmic reticulum fractions. Comparison of polyoma virus-transformed and normal baby-hamster kidney cells reveals some interesting differences in their microsomal fractionation patterns and the characteristics of the Na+/K+-Mg2+ adenosine triphosphatase of their surface membranes, in particular a tenfold lower Km in the virus-transformed cells. The fractionation patterns of normal and spontaneously transformed rat embryo fibroblasts are also briefly discussed, particularly in relation to the significance of the observation that both the surface membrane and endoplasmic reticulum from these cells can be subfractionated.
Kinetic studies of nitrogenase from soya-bean root-nodule bacteroids
Authors: Bergersen F. J. , Turner G. L.
Published: August 2015

The apparent Michaelis constants [K′(N2) and K′(C2H2)] and the corresponding apparent maximum velocity values (V′) for soya-bean bacteroid nitrogenase increased concomitantly in response to increases in nitrogenase Fe–protein concentration and ATP concentration in cell-free assays and in response to O2 pressure in intact nodules and bacteroid suspensions. K′(C2H2) in cell-free assays was also affected by pH and by Na2S2O4 concentration. Nitrogenase Fe–protein behaved as a catalytic effector reacting at interacting sites on the nitrogenase Fe–Mo–protein. The results indicated that the Fe–Mo–protein probably bears the catalytic sites for N2 and C2H2 reduction. It is concluded that reduction of N2 or C2H2 by this nitrogenase involves a reaction mechanism with a sequence of unknown order. The sequence in which substrate, enzyme, effector, ATP and reductant react determines which of the various rate-constants are involved in the apparent Michaelis constant, whose true kinetic meaning was thus unresolved.
A kinetic study of rabbit muscle pyruvate kinase
Authors: Ainsworth S. , Macfarlane N.
Published: August 2015

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg2+ catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg2+, pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg2+, form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.
The effect of l-ascorbate on catecholamine biosynthesis (Short Communication)
Authors: Stone K. J. , Townsley B. H.
Published: August 2015

l-Ascorbate stimulates the enzymic hydroxylation of phenylalanine in vitro by recycling tetrahydrobiopterin, which reduces O2 utilized in the reaction. It is suggested that ascorbate might have a similar function in vivo; this would explain the apparent regulation of tyrosine hydroxylase and tryptophan hydroxylase activities by this vitamin.
The chemistry of the collagen cross-links. Age-related changes in the reducible components of intact bovine collagen fibres
Authors: Robins Simon P. , Shimokomaki Massami , Bailey Allen J.
Published: August 2015

The change in the amounts of the three major reducible cross-links was followed throughout the bovine-life span. The major reducible cross-link in embryonic skin is 6,7-dehydro-Nε -(2-hydroxy-5-amino-5-carboxypentyl)hydroxylysine, but this is gradually replaced in the latter stages of gestation or early postnatal growth period by two other Schiff bases, 6,7-dehydro-Nε-(5-amino-5-carboxypentyl)hydroxylysine and a component not yet identified, designated Fraction C. These latter two Schiff bases increase in amount during the rapid growth period to a maximum, after which they then slowly decrease until at maturity they are virtually absent. The proportion of these Schiff bases closely reflects the rate of growth, i.e. the amount of newly synthesized collagen present at any one time. Similarly, the three Schiff bases present in tendon and the one in cartilage slowly decrease during maturation. No evidence for the possible stabilization of these aldimine bonds during maturation by reduction in vivo was found by three different analytical techniques. Concurrently with the decrease in the proportion of the Schiff bases some new reducible components increased during maturation, but their characterization as Nε-glycosylamines demonstrated that they were not related to the lysine-derived aldehyde components. The significance of these components in the aging process cannot at present be assessed. As no evidence was obtained for any new reducible cross-links replacing the Schiff bases, it is probable that the latter are intermediate cross-links and that during maturation they are stabilized to some as yet unknown non-reducible cross-link as previously proposed (Bailey, 1968).
Bone collagen metabolism in vitamin D deficiency (Short Communication)
Authors: Barnes M. J. , Constable B. J. , Morton L. F. , Kodicek E.
Published: August 2015

Radioisotope studies have shown that collagen synthesized in the shafts of bones from rachitic rats and chicks is similar in chain composition to that normally synthesized. However, the extent of lysine hydroxylation in both α1- and α2-chains is increased, by approx. 15–30% in the rat and by 50% in the chick.
The synthesis of medium-chain fatty acids by lactating-rabbit mammary gland studied in vitro (Short Communication)
Authors: Strong Christopher R. , Carey Eric M. , Dils Raymond
Published: August 2015

The proportion of C8:0 and C10:0 fatty acids synthesized by the microsomal plus particle-free supernatant fraction from lactating rabbit mammary gland is enhanced at high protein concentrations. This fraction appears to contain a soluble high-molecular-weight factor that modifies the specificity of the fatty acid synthetase complex for termination of the growing acyl chain.
Studies of nuclei separated by zonal centrifugation from liver of rats treated with thioacetamide
Authors: Gonzalez-Mujica F. , Mathias A. P.
Published: August 2015

1. The effects of the inclusion of thioacetamide in the diet on the properties of rat liver nuclei were studied both in adolescent rats, in which the parenchymal cells contain diploid nuclei, and in young adult rats, with a high proportion of tetraploid nuclei. 2. These investigations included a survey of the sedimentation properties of the nuclei, the nuclear volumes, content of DNA, RNA and protein, the incorporation in vivo of [3H]thymidine into DNA and [14C]orotate into RNA, and measurements of the activity of RNA polymerase and ribonuclease. These studies were conducted on nuclei fractionated by zonal centrifugation. 3. In both groups of animals, exposure to thioacetamide produced large numbers of nuclei that were abnormal in their chemical composition and enzymic activity. The changes were complex as regards both the types of nuclei that were affected and in their variation with time. 4. In adolescent rats two waves of synthesis of DNA and RNA were observed, one at 3 days and the other after 2 weeks of treatment. The first decline in the incorporations into both DNA and RNA coincided with a decrease in the pool sizes of some of the precursors. The activity of RNA polymerase was not substantially altered. A marked increase in the content of protein was observed before the first wave of synthesis. The normal progressive increase in tetraploid nuclei was prevented. 5. In young adult rats two waves of DNA synthesis were detected. Each was preceded by a large increase in the amount of protein per nucleus but was not accompanied by increased RNA synthesis. After 4 weeks of treatment, the diploid stromal nuclei appeared mainly unaffected and large numbers of tetraploid nuclei with a greatly increased quantity of protein were observed.
Cell-free protein synthesis in heart and skeletal muscles from polymyopathic hamsters
Authors: Bester André J. , Gevers Wieland
Published: August 2015

1. Cell-free protein synthesis was studied in striated and smooth muscles in an attempt to elucidate the primary genetic defect in polymyopathic hamsters. 2. When washed membrane-free polyribosomes from myopathic and control heart muscle were individually recombined with pH5 enzymes from both types of animals, the pH5 enzymes from myopathic muscle were less active in polypeptide synthesis than those from controls, irrespective of the source of polyribosomes. 3. The same defect was present in skeletal-muscle preparations. 4. Both the initial rate and the maximum extent of incorporation were affected in the defective preparations from myopathic muscle. 5. Concentration differences, with respect to total protein and RNA, were not responsible. 6. Preincubation of the pH5 enzymes resulted in a greater degree of inhibition. 7. The defect in the pH5 enzymes from myopathic muscle was also expressed in poly(U)-directed polyphenylalanine synthesis. 8. Acid proteinase activity in extracts of control and myopathic muscle was the same but general ribonuclease activity in the latter extracts was higher. 9. The defect was also present when both types of pH5 enzymes were prepared in the presence of the ribonuclease-asborbent bentonite. 10. pH5 enzymes from uterine smooth muscle, brains and livers of myopathic animals were similarly affected in homologous and heterologous combinations. 11. It is concluded that the general tissue defect is both qualitative and quantitative in nature, implying that there is a shortage of some essential soluble component in the pH5 fraction which is accompanied by the presence of an altered substituent. This prevents the attainment of extents of polypeptide synthesis in vitro obtained in control extracts from unaffected animals.
Turnover of protein-bound serine phosphate in respiring slices of guinea-pig cerebral cortex. Effects of putative transmitters, tetrodotoxin and other agents
Authors: Reddington Martin , Rodnight Richard , Williams Michael
Published: August 2015

1. The effect of various agents on the turnover of protein-bound phosphorus in respiring slices of cerebral cortex was studied. 2. Confirming previous work turnover was increased by the application of electrical pulses for 10s to the tissue. 3. Turnover was also increased by exposure of the slices for 10min to noradrenaline (0.5mm), 5-hydroxytryptamine (1μm) and histamine (0.1mm). 4. When slices were stimulated by electrical pulses in the presence of histamine the increase in turnover was the sum of the responses given by each agent above, suggesting that different phosphorylating systems were involved. 5. Tetrodotoxin (0.5μm) blocked the increased turnover due to electrical pulses, but not that due to histamine. Tetrodotoxin also prevented the increase in tissue cyclic AMP content caused by the application of electrical pulses. 6. Phosphoprotein turnover was not affected by adenosine, despite the increase in tissue cyclic AMP content given by this agent. 7. Adenosine blocked the phosphoprotein response to histamine, but did not affect the response to electrical pulses. 8. The results are discussed in relation to the hypothesis that the stimulation of protein phosphorus turnover by electrical pulses is secondary to the release of cyclic AMP in the tissue.
Insulin and the regulation of adipose-tissue acetyl-coenzyme A carboxylase
Authors: Halestrap Andrew P. , Denton Richard M.
Published: August 2015

Rat epididymal fat-pads were incubated for 30min with glucose (2mg/ml) in the presence or absence of insulin. A twofold or greater increase in acetyl-CoA carboxylase activity was observed in extracts from insulin-treated tissue provided that assays were performed rapidly after extraction. This effect of insulin was evident whether or not extracts were prepared with albumin, and was not noticeably diminished by the presence of citrate or albumin or both in the assay. Incubation of extracts before assay led to activation of acetyl-CoA carboxylase and a marked diminution in the insulin effect. The enzyme in extracts was very sensitive to reversible inhibition by palmitoyl-CoA even in the presence of albumin (10mg/ml); inhibition persisted on dilution of enzyme and inhibitor. It is suggested that the observed activation of acetyl-CoA carboxylase by insulin may reflect changes in enzyme activity in the fat-cell resulting from the reduction of long-chain fatty-acyl-CoA that occurs in the presence of insulin. Activation of the enzyme with loss of the insulin effect on incubation of the extracts may be due to the slow dissociation of long-chain fatty acyl-CoA from the enzyme.
Control of phosphoenolpyruvate synthesis in guinea-pig mitochondria
Authors: Wilson M. B.
Published: August 2015

1. The synthesis of phosphoenolpyruvate and the O2 consumption from the tricarboxylic acid-cycle intermediates citrate, α-oxoglutarate, malate and succinate by guinea-pig mitochondria were compared. Malate was the most effective of these precursors; there was no synthesis of phosphoenolpyruvate from succinate. 2. The addition of palmitate, acetoacetate and ATP enhanced the synthesis of phosphoenolpyruvate from citrate and α-oxoglutarate. Palmitate and ATP increased the O2 consumption, whereas acetoacetate had no effect on this parameter. 3. Octanoate depressed the synthesis of phosphoenolpyruvate from citrate, α-oxoglutarate and malate and increased the O2 consumption. Pentenoic acid had no effect on phosphoenolpyruvate synthesis from any of the substrates used, although it increased the uptake of O2. These findings might be relevant to the control of gluconeogenesis in vivo.
Biosynthesis of spheroidene and hydroxyspheroidene in Rhodopseudomonas species: experiments with nicotine as inhibitor (Short Communication)
Authors: Singh R. K. , Ben-Aziz A. , Britton G. , Goodwin T. W.
Published: August 2015

Neurosporene replaces spheroidene and hydroxyspheroidene as the main carotenoid in Rhodopseudomonas spheroides and Rhodopseudomonas gelatinosa grown in the presence of nicotine. On removal of the nicotine, spheroidene and hydroxyspheroidene are formed at the expense of the accumulated neurosporene. This shows that nicotine inhibits introduction of the C-1 tertiary hydroxyl groups, and supports the postulated pathway neurosporene→spheroidene→hydroxyspheroidene.
Interrelationships in trace-element metabolism in metal toxicities in a cobalt-resistant strain of Neurospora crassa
Authors: Venkateswerlu G. , Sastry K. Sivarama
Published: August 2015

A strain of Neurospora crassa was isolated by training the mould to grow on media containing high concentrations of Co2+. This strain, the CoR strain, exhibited approximately tenfold the resistance of the parent strain to Co2+ and Ni2+ but not to Zn2+ or Cu2+. Co2+ toxicity in the CoR strain was reversed by Mg2+ but not by Fe3+. Also, Co2+ did not affect iron metabolism in this strain. It is suggested that the mechanism of resistance in the CoR strain involves an alteration in the pattern of iron metabolism such that the latter is no longer adversely affected by toxic concentrations of Co2+. The CoR strain is genetically stable and is most probably a result of a resistance mutation in N. crassa induced by Co2+.
3,4-Dihydroxyphenylalanine (dopa) decarboxylase activity in the arthropod nervous system
Authors: Murdock L. L. , Wirtz R. A. , Köhler G.
Published: August 2015

1. When homogenates of brains from mature adult locusts (Locusta migratoria) were incubated with l-3-(3,4-dihydroxyphenyl)[3-14C]alanine the major radioactive metabolite was dopamine, suggesting the presence of a dopa (3,4-dihydroxyphenylalanine) decarboxylase. 2. Decarboxylation of l-dopa by this tissue, measured under optimum conditions by a radiochemical method, was 21μmol of CO2/h per g wet wt. Apparent decarboxylation of l-tyrosine proceeded at 0.34μmol of CO2/h per g wet wt. There was no detectable decarboxylation of l-tryptophan, l-histidine or l-phenylalanine. 3. Dopa decarboxylase activity was found in all major regions of the ventral nerve cord of the mature locust (range: 4–7μmol of CO2/h per g wet wt.) but was low or absent in thoracic peripheral nerve. 4. Marked decarboxylation of l-dopa was found in homogenates of brains of four other species of insects, and in brain and ventral nerve cord, but not in the claw nerve, of the crayfish. 5. The activity of the locust brain enzyme may be slightly lower at the time of imaginal ecdysis than during the mature period. By contrast, the dopa decarboxylase that produces dopamine as an intermediate in cuticle biosynthesis is known to be high in activity at the time of ecdysis and low in activity during the intermoult stages.
Microbial metabolism of C1 and C2 compounds. The role of acetate during growth of Pseudomonas AM1 on C1 compounds, ethanol and β-hydroxybutyrate
Authors: Dunstan Patricia M. , Anthony C.
Published: August 2015

Pseudomonas AM1 grows on β-hydroxybutyrate and methanol at similar rates. β-Hydroxybutyrate is not metabolized by way of the glyoxylate bypass, but is assimilated by the novel route (with acetate as an intermediate) that operates during growth of this organism on ethanol. Evidence from short-term labelling experiments indicates that acetate, which is a possible intermediate in the assimilation of C1 compounds, is rapidly metabolized to glycine during growth of Pseudomonas AM1 on methanol.
The molecular weight and thiol residues of acetyl-coenzyme A synthetase from ox heart mitochondria
Authors: Londesborough John C. , Yuan Sung Ling , Webster Leslie T.
Published: August 2015

1. A constant molecular weight of 57000 was obtained by gel filtration of highly purified acetyl-CoA synthetase over a 1000-fold range of enzyme concentrations. The amino acid analysis is reported. 2. With native enzyme at 20°C the relatively rapid reaction of four thiol residues with p-hydroxymercuribenzoate caused an immediate inhibition reversible by either CoA or mercaptoethanol. Other substrates did not protect against this rapid inhibition. 3. The much slower reaction of the remaining four thiol residues was independent of the concentration of the mercurial, first-order with respect to enzyme, and had a large energy of activation (+136kJ/mol), suggesting that a conformation change in the protein was rate-limiting. This slow phase of the reaction was accompanied by an irreversible inactivation of the enzyme. 4. The effects of substrates on this irreversible inactivation at pH7.0 in 5 mm-MgCl2 indicated strong binding of ATP and pyrophosphate by the enzyme (concentrations for half-maximal effects, K½, were <30μm and <10μm respectively) and weaker binding of acetyl-CoA (K½ about 1 mm), AMP (K½ about 2mm) and acetate. In the presence of acetate, MgCl2 and p-hydroxymercuribenzoate, titration of the enzyme with ATP revealed at least two ATP binding sites/mol. 5. The experiments suggest that reaction of the thiol residues with mercurial causes loss of enzymic activity by altering the structure of the enzyme, rather than that the thiol residues play a direct role in the catalysis.
The distinction between γ-glutamylhydroxamate synthetase and l-glutamine–hydroxylamine glutamyltransferase activities in rat tissues. Studies in vivo
Authors: Herzfeld Annemarie , Estes Nathan A.
Published: August 2015

The formation of γ-glutamylhydroxamate by homogenates under optimum assay condition showed an inconstancy in the ratios of the enzyme activities utilizing l-glutamate and ATP (γ-glutamylhydroxamate synthetase) and l-glutamine and ADP (l-glutamine–hydroxylamine glutamyltransferase) in a number of normal and neoplastic rat tissues. Although γ-glutamylhydroxamate synthetase activities in adult livers and kidneys were identical in males and females, l-glutamine–hydroxylamine glutamyltransferase activities in the organs of females were significantly lower. The developmental formations of the two activities in liver, kidney, brain and muscle were not simultaneous. The l-glutamine–hydroxylamine glutamyltransferase activity in foetal liver or neonatal kidney could be prematurely evoked by thyroxine, but the γ-glutamylhydroxamate synthetase activity remained unchanged. Injections of cortisol also had dissimilar effects on the two activities in thymus and hepatomas. The discrepant tissue distribution, asynchronous developmental formation and differential response to several hormonal stimuli provide evidence in vivo that the two activities are not catalysed by the same protein.
The role of magnesium ions in β-galactosidase-catalysed hydrolyses. Studies on charge and shape of the β-galactopyranosyl-binding site
Authors: Case Gillian S. , Sinnott Michael L. , Tenu Jean-Pierre
Published: August 2015

1. β-d-Galactopyranosyl trimethylammonium bromide is a competitive inhibitor of β-galactosidase, Ki=1.4±0.2mm at 25°C. 2. Tetramethylammonium bromide is not an inhibitor (Ki>0.2m). 3. The kinetics of deactivation of Mg2+-saturated, and of inhibitor-and Mg2+-saturated, enzyme in 10mm-EDTA are similar. 4. The apparent Ki for the glycosylammonium salt is approx. 2.2mm in the absence of Mg2+. 5. It is therefore concluded that Mg2+ and the inhibitor bind independently, and that the Mg2+ does not act as an electrophilic catalyst. 6. Complexant fluorescence measurements indicate binding of 1 Mg2+ ion per 135000-dalton protomer. 7. This stoicheiometry is confirmed by equilibrium dialysis. 8. 1,6-Anhydrogalactopyranose is neither a substrate (kcat./Km< 3×10-2m-1·S-1) nor an inhibitor (Ki>0.2m). 9. Considerations of conformations available to the cationic inhibitor and to the anhydrogalactose indicate that the substrate is bound with the pyranose ring in a conformation not greatly different from the normal chair (C1) conformation.
Partial purification and properties of the common inherited forms of adenosine deaminase from human erythrocytes
Authors: Osborne W. R. A. , Spencer N.
Published: August 2015

1. The partial purification of adenosine deaminase, types 1, 2 and 2–1, from human erythrocytes is described. 2. The isoenzyme components characteristic of the three forms of the enzyme were partially resolved by chromatography on DEAE-Sephadex. 3. Gel chromatography of the various forms of the enzyme gave estimates of the molecular weights in the range 30000–35000. 4. Electrophoresis in starch gels containing increasing percentages of starch did not reveal any differences in molecular weight between the genetic variants or their isoenzyme components. 5. Analytical isoelectric-focusing experiments in polyacrylamide gels gave the following pI values for the four isoenzyme components present in type 2–1 erythrocytes: 4.70, 4.83, 4.94 and 5.06. 6. All forms of the enzyme gave Km values for adenosine of about 30μm and Ki values of about 8μm for the competitive inhibitor purine riboside. 7. Reaction rates of the type 1 and 2 enzymes were measured at different temperatures. Both enzymes gave values for the energy of activation for hydrolysis of adenosine of about 33.4kJ/mol (8kcal/mol). 8. Heat inactivation of all forms of the enzyme was markedly dependent on ionic strength, the rate of inactivation increasing with increasing ionic strength. The type 1 and type 2 forms of the enzyme differed significantly in their susceptibility to heat inactivation. From the variation of rates of inactivation with temperature, values were obtained for the energies of activation for the heat inactivation of both enzymes as follows: type 1 enzyme 275.5kJ/mol (65.9kcal/mol) and type 2 enzyme 241.6kJ/mol (57.8kcal/mol.).
The amino acid sequence of cytochrome c from Nigella damascena L. (love-in-a-mist)
Authors: Brown R. H. , Boulter D.
Published: August 2015

The amino acid sequence of cytochrome c from Nigella damascena L. was determined on 0.2μmol of protein. Peptides from a single chymotryptic digest were analysed by the dansyl–Edman procedure. These peptides were ordered by reference to the sequences of other plant cytochromes c, assuming that the Nigella cytochrome is homologous with the other cytochromes. Many of the Nigella peptides were one or two residues short when compared with the corresponding chymotryptic peptides from other plant cytochromes c. These residues are assumed to have been removed by an endogenous carboxypeptidase, and the identification and placing of these residues is entirely based on homology. These residues are numbered 3, 18, 42, 43, 44, 54, 67, 72, 73, 82 and 105. A number of other positions are almost entirely placed by homology. These are positions which could not be placed definitely by dansyl–Edman analysis or by dansylation after digestion with carboxypeptidase A, and are numbered 14, 15, 16, 39, 40, 85, 86, 87 and 88. Except for residue 15, all residues based entirely, or nearly so, on homology have been previously found invariant in sequences of plant cytochromes c. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50017, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.
The formation of a β-(1→4)-d-galactan chain catalysed by a Phaseolus aureus enzyme
Authors: Panayotatos N. , Villemez C. L.
Published: August 2015

With a particulate enzyme preparation from Phaseolus aureus hypocotyls, UDP-α-d-[U-14C]galactose served as a precursor for a number of products. One of these products was characterized as a β-(1→4)-linked galactan. The ADP-, GDP-, TDP- and CDP- derivatives of α-d-galactose did not serve as biosynthetic precursors for any products insoluble in 70% ethanol, nor as substrates for a sugar nucleotide 4-epimerase which is present in the particulate enzyme preparation. The 14C-labelled β-(1→4)-galactan is alkali-insoluble and was characterized by analysis of partial acetolysis products. The labelling pattern of the [14C]oligosaccharides derived from acetolysis indicates that (1) only slightly more than two [14C]galactose moieties are added to the growing polysaccharide chain on average, and (2) these additions take place at the reducing end of the polysaccharide chain. The radioactive β-(1→4)-linked galactan chain represented 8.5% of the radioactivity initially added, and 20% of the water- and butanol-insoluble products derived from UDP-α-d-[14C]galactose. Total hydrolysis of the alkali-insoluble fraction of Phaseolus aureus hypocotyl yielded d-glucose and d-mannose in a 5:1 ratio but no detectable quantities of d-galactose. A trace quantity of a radioactive disaccharide, identified as (1→3)-linked galactobiose, was isolated from the partial acetolysate of the alkali-insoluble [14C]polysaccharide material. Also isolated from this partial acetolysate was a C-1 derivative of [14C]galactose, which could not be identified. An alkali-soluble galactose-containing polysaccharide was also synthesized in this enzymic reaction, and represented 20% of the water- and butanol-insoluble products derived from UDP-α-d-[14C]galactose. The spectrum of radioactive oligosaccharides produced by partial acetolysis of this alkali-soluble polysaccharide material was different from that obtained from the alkali-insoluble polysaccharide, indicating a different structure.
Multiple aggregation factors in cartilage proteoglycan (Short Communication)
Authors: Gregory J. D.
Published: August 2015

Proteoglycan as isolated from bovine nasal septa is a partially aggregated complex containing linking factors. Two essential factors can be separated from proteoglycan subunits and resolved in a CsCl density gradient. Chondroitinase digestion of the purified proteoglycan subunits does not interfere with their aggregation when the linking factors are added.
A comparative study of aldolase from human muscle and liver
Authors: Eagles Peter A. M. , Iqbal Muzaffar
Published: August 2015

Aldolase was purified from human skeletal muscle and human liver by techniques capable of processing large quantities (10–20kg) of tissue. The methods used also proved convenient for isolating aldolase on a large scale from other mammalian and avian sources. Aldolase from both human liver and muscle was crystallized; each gave two crystalline forms, depending on the conditions of crystallization. X-ray studies on the muscle aldolase crystals suggest a close structural similarity between human and rabbit muscle aldolase. Aldolases from human muscle and liver have similar pH optima and pH stability but their stability to heat treatment differs. The effect of heat on the enzymes may therefore provide an easy means of distinguishing them. The kinetic constants Km and kcat. for these aldolases are similar to other mammalian aldolases. Amino acid analyses and tryptic peptide ‘mapping’ show that the primary structures of the two aldolases differ greatly.
The alkaline cleavage and borohydride reduction of cartilage proteoglycan
Authors: Robinson H. Clem , Hopwood John J.
Published: August 2015

A method for the rapid isolation and purification of proteoglycan by using neutral solutions of LiBr for extraction and density-gradient centrifugation is described. The effect of 0.5m-KOH on isolated proteoglycan has been studied by using NaB3H4 to reduce and label the chondroitin sulphate chains released. This study has established: (a) that at least 95% of the chondroitin sulphate chains are attached to the proteoglycan by alkali-labile bonds between xylose and serine; (b) that random degradation of the chondroitin sulphate chains does not occur to any significant extent; (c) that the method is convenient for the determination of polysaccharide number-average molecular weights.
Effects of protein-modifying reagents on an isoenzyme of potato apyrase
Authors: Valenzuela M. Antonieta , Del Campo Guillermo , Marín Eugenio , Traverso-Cori Aída
Published: August 2015

Treatment of an isoenzyme of potato apyrase of high adenosine triphosphatase/adenosine diphosphatase (ATPase/ADPase) ratio with iodine, N-acetylimidazole or tetranitromethane inactivates the ATPase activity of this enzyme faster than its ADPase activity. There was protection by substrates with the two last-named substances. This and the appearance of nitrotyrosine suggests the participation of tyrosyl residues in both enzymic activities of potato apyrase. The participation of thiol groups is excluded by the insensitivity of apyrase to p-chloromercuribenzoate. Also, 2-hydroxy-5-nitrobenzyl bromide or carboxymethylation produce the same rate of inactivation of ATPase and ADPase activities. Substrates protect both activities from inactivation. Hydrogen peroxide and photo-oxidation inactivate ATPase activity faster than ADPase activity. There is no protection by substrates. Analysis of pH effects on Vmax. and Km suggest different pK values for the amino acid residues at the ATP and ADP sites.
Studies with a reconstituted muscle glycolytic system. The rate and extent of creatine phosphorylation by anaerobic glycolysis
Authors: Scopes Robert K.
Published: August 2015

A mixture of purified muscle glycolytic enzymes was reconstituted and the mixture shown to behave in a fashion analogous to that occurring in vivo. Glycolysis leads to ATP production in muscle and results in the phosphorylation of creatine. The extent of this phosphorylation by anaerobic glycolysis was shown to depend to a small extent on the relative proportions of available Pi and creatine initially, but more importantly on the first step in glycolysis, in this case the enzyme phosphorylase. With less than 0.1% of the phosphorylase in the a form, only about one-third of the creatine was phosphorylated in 30min, whereas with 4% or more of phosphorylase a, 90% of the creatine was phosphorylated within this time. Inclusion of an adenosine triphosphatase decreased the steady-state concentration of phosphocreatine in the system. Calculations of the theoretical concentrations of ADP and AMP showed that phosphorylase b was almost inactive even in the presence of 9μm-AMP, because of ATP inhibition. With phosphorylase a present, glycolysis was able to continue at least until the calculated concentration of MgADP− was only 7μm, and AMP in the sub-μmolar range. The relation of these values to measured concentrations of nucleotides and to phosphorylase a percentages in intact muscle is discussed.
Control of the production of exo-β-N-acetylglucosaminidase by Bacillus subtilis B. Derepression during gluconeogenesis and initial stages of sporulation
Authors: Brewer Stephen J. , Berkeley Roger C. W.
Published: August 2015

1. The control of exo-β-N-acetylglucosaminidase (EC 3.2.1.30) production by Bacillus subtilis B growing on a chemically defined medium was studied. 2. The enzyme was repressed during exponential growth by those carbon sources that enter the glycolytic pathway above the level of phosphoenolpyruvate. When exponential growth ceased as a result of low concentrations of the nitrogen, carbon or metal ion components of the medium, the enzyme was formed and its amount could be increased by the addition of cell-wall fragments as inducer. 3. The enzyme was de-repressed and could be induced during exponential growth on non-glycolytic compounds metabolized directly into pyruvate, acetyl-CoA or tricarboxylic acid cycle intermediates. 4. The major difference in the metabolism of the organism utilizing these two groups of compound was the existence of high activities of phosphoenolpyruvate carboxylase required for gluconeogenesis. 5. It is concluded that the de-repression of glucosaminidase occurs when the only principal change detected in the intermediary metabolism of the organism was the presence of high activities of phosphoenolpyruvate carboxylase. 6. When the organism was grown on media containing repressing compounds, the enzyme was only de-repressed on entry of the cells into the initial stages of sporulation, where phosphoenolpyruvate carboxylase activity, even in the presence of excess of glucose, increased in parallel with glucosaminidase, neutral proteinase and alkaline phosphatase activities. 7. These results suggest a strong link, at the level of the tricarboxylic acid cycle, between the control of phosphoenolpyruvate carboxylase and the control of the de-repression of glucosaminidase and sporulation.
The nature of the acid-volatile selenium in the liver of the male rat
Authors: Diplock A. T. , Caygill Christine P. J. , Jeffery Elizabeth H. , Thomas C.
Published: August 2015

1. The properties of rat liver acid-volatile selenium have been compared with those of H2Se and (CH3)2Se. 2. In model experiments oxidation-sensitive H275Se was trapped quantitatively under anaerobic conditions in 0.1m-AgNO3, and (CH3)275Se was trapped quantitatively in 8m-HNO3. The acid-labile selenium of a liver homogenate, and of a microsomal fraction, was found to behave quite unlike (CH3)275Se and in a manner indistinguishable from H275Se. 3. It was concluded that the acid-volatile material is certainly not (CH3)2Se and that it is probably H2Se. 4. The significance of these findings is discussed in relation to current knowledge about the metabolism and detoxication of selenium, and a scheme is proposed which incorporates this knowledge with recent observations on the interactions between trace amounts of selenium and tocopherol, and the production of acute selenium deficiency by Ag+ in vitamin E-deficient rats.
Comparative studies on the carbohydrate-containing membrane components of normal and adenosine 3′:5′-cyclic monophosphate-treated Chinese-hamster ovary cells
Authors: Baig M. Mansoor , Roberts R. M.
Published: August 2015

1. We investigated some of the changes in plasma-membrane composition that accompany the alteration in cell growth and morphology induced by treating Chinese-hamster ovary cells with dibutyryladenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP). 2. A double-labelling technique was employed in which normal cells were given 14C-labelled precursor, and those treated with dibutyryl cyclic AMP were given 3H label. l-Leucine, d-glucosamine, and l-fucose were used to label the membranes. 3. After 3 days growth, the two populations of cells were harvested by trypsin treatment, the cells were pooled, and plasma membranes isolated. Proteins and glycoproteins of the membranes were separated by electrophoresis on sodium dodecyl sulphate–polyacrylamide gels, and the radioactive profiles for 14C and 3H and the staining patterns with Amido Black were compared. 4. Although certain components of the membrane from treated cells showed marked quantitative changes, there was neither major addition nor major deletions of components. 5. Complete proteolysis of the mixed membranes, of the material released from the cell surface by trypsin, and of the glycoproteins released from the cells into the medium, gave a series of radioactive glycopeptides when either fucose or glucosamine was employed as precursor. 6. After such glycopeptides were fractionated on columns of Sephadex G-50, marked differences in the elution profiles of 3H and 14C were noted. Dibutyryl cyclic AMP evidently causes alterations in the overall composition of the carbohydrate components of the cell surface. It was not possible to decide whether this was solely the result of the same glycoproteins being formed but in different proportions, or the result of modifications of oligosaccharide side chains on some of the glycoproteins. 7. Some of the changes were not unlike the reverse of those that accompany the transformation of fibroblasts by oncogenic viruses, and our results lend credence to the idea that the lowered amount of cyclic AMP noted in transformed cells is responsible for their altered surface properties.
Metabolic alterations produced in the liver by chronic ethanol administration. Comparison between the effects produced by ethanol and by thyroid hormones
Authors: Israel Y. , Videla L. , Macdonald A. , Bernstein J.
Published: August 2015

1. Liver slices from rats treated with thyroxine show an increased rate of O2 consumption. The extra consumption, but not the basal respiration, can be abolished by ouabain. 2. Dinitrophenol is not effective in increasing the rate of O2 consumption of liver slices from thyroxine-treated animals but its effectiveness can be recovered in the presence of ouabain. 3. (Na++K+)-stimulated adenosine triphosphatase activity of liver was increased by administration of thyroxine in vivo. No changes were found in total Mg2+-stimulated adenosine triphosphatase activity. 4. Mitochondrial α-glycerophosphate dehydrogenase and microsomal NADPH oxidase activity were increased by both thyroxine and chronic ethanol treatment. 5. Liver slices from animals chronically treated with ethanol synthesize urea at an increased rate. 6. Mitochondrial size (section area) is markedly increased in the liver of animals chronically treated with ethanol. 7. Acute administration of ethanol in doses of 4 and 6g/kg significantly increases the uptake of 131I-labelled thyroxine by the liver. 8. Work reported here, along with results from other investigators, indicates marked similarities between the effects produced in the liver by chronic administration of ethanol and by thyroid hormones.
Normalization in the fitting of data by iterative methods. Application to tracer kinetics and enzyme kinetics
Authors: Ottaway J. H.
Published: August 2015

1. The normalization of biochemical data to weight them appropriately for parameter estimation is considered, with reference particularly to data from tracer kinetics and enzyme kinetics. If the data are in replicate, it is recommended that the sum of squared deviations for each experimental variable at each time or concentration point is divided by the local variance at that point. 2. If there is only one observation for each variable at each sampling point, normalization may still be required if the observations cover more than one order of magnitude, but there is no absolute criterion for judging the effect of the weighting that is produced. The goodness of fit that is produced by minimizing the weighted sum of squares of deviations must be judged subjectively. It is suggested that the goodness of fit may be regarded as satisfactory if the data points are distributed uniformly on either side of the fitted curve. A chi-square test may be used to decide whether the distribution is abnormal. The proportion of the residual variance associated with points on one or other side of the fitted curve may also be taken into account, because this gives an indication of the sensitivity of the residual variance to movement of the curve away from particular data points. These criteria for judging the effect of weighting are only valid if the model equation may reasonably be expected to apply to all the data points. 3. On this basis, normalizing by dividing the deviation for each data point by the experimental observation or by the equivalent value calculated by the model equation may both be shown to produce a consistent bias for numerically small observations, the former biasing the curve towards the smallest observations, the latter tending to produce a curve that is above the numerically smaller data points. It was found that dividing each deviation by the mean of observed and calculated variable appropriate to it produces a weighting that is fairly free from bias as judged by the criteria mentioned above. This normalization factor was tested on published data from both tracer kinetics and enzyme kinetics.