bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin biosynthesis in the bullhead, Ictalurus nebulosus, and the effect of temperature
Authors: Moule Margaret L. , Yip Cecil C.
Published: August 2015

Insulin biosynthesis in the brown bullhead, Ictalurus nebulosus (Le Sueur), was studied by measuring the incorporation in vitro of [3H]leucine into proteins of the principal islet. The tissue was incubated for 6–15h in Krebs–Ringer bicarbonate buffer with [3H]leucine, supplemented with amino acids and glucose. Proteins, precipitated with trichloroacetic acid and extracted with acid ethanol, were separated by gel-filtration on Biogel P-30 in 3m-acetic acid. Three major components were found after incubation of the islets at 22°C. On the basis of the results of sulphitolysis, biological activity and the demonstrated precursor–product relationship, components I and II were identified as proinsulin and insulin respectively. The third component was not identified. At 12°C, [3H]leucine was incorporated only into proinsulin. No radioactivity was found in insulin or the unidentified component III at 12°C as was found after incubation at 22°C. When the temperature was lowered from 22° to 12°C after 3h of a 15h incubation, decreased conversion of proinsulin into insulin resulted at the lower temperature compared with the control tissue maintained at 22°C. When the temperature was raised from 12° to 22°C at 3h of a 15h incubation, conversion of proinsulin into insulin occurred. No conversion occurred in the control tissue with the temperature maintained at 12°C. No qualitative difference in the incorporation of [3H]leucine into proinsulin and its conversion into insulin at 12° and 22°C could be demonstrated between islet tissue from fish acclimated to less than 12°C or to 22°C. The results suggest that the enzyme(s) responsible for converting proinsulin into insulin in the bullhead may be temperature sensitive with low activity at 12°C.
Inhibition of phosphatidylcholine synthesis in mammary tissue by 2-chloroethyltrimethylammonium chloride (Short Communication)
Authors: Infante J. P. , Kinsella J. E.
Published: August 2015

The incorporation of choline into phosphatidylcholine in mammary tissue from lactating cows is impeded by 2-chloroethyltrimethylammonium chloride at concentrations ranging from 0.3 to 9mm. This compound is a non-competitive inhibitor of choline kinase (Km 0.25mm), having Ki 17mm. It is suggested that 2-chloroethyltrimethylammonium chloride may inhibit choline entry into the tissue.
The metabolism of acetamidothiazoles in the rat. 2-Acetamido-, 2-acetamido-4-methyl- and 2-acetamido-4-phenyl-thiazole
Authors: Chatfield D. H. , Hunter W. H.
Published: August 2015

The metabolism of some anti-inflammatory acetamidothiazoles was studied in the rat. The main metabolites were the corresponding acetylthiohydantoic acids, produced by fission of the thiazole ring. Minor metabolites arising from oxidation of the methyl or phenyl substituents were also identified. The structures of metabolites were established spectroscopically (u.v., i.r., n.m.r. and mass spectroscopy) and by identification with authentic specimens. The excretion of the original compounds and of metabolites, labelled with 14C is also reported.
Effect of insulin on pyruvate metabolism in epididymal adipose tissue of the rat. Correlation of intracellular pyruvate contents and pyruvate dehydrogenase activity
Authors: Berman Bruce G. , Halperin Mitchell L.
Published: August 2015

A method is described to measure the intracellular content of pyruvate and lactate in epididymal adipose tissue of the rat. The intracellular pyruvate concentration was approx. 330μm. Intracellular pyruvate contents and the rates of pyruvate output were increased when NNN′N′-tetramethyl-p-phenylenediamine was added, and decreased in the presence of alanine. Insulin addition caused an increase in intracellular pyruvate contents only at the earlier time-period studied (1.5min as against 20min). Pyruvate dehydrogenase activity was increased in adipose tissue incubated in vitro with insulin. This increase occurred subsequent to the rise in the intracellular pyruvate content induced by insulin addition. The possible physiological implications are discussed.
Reconstitution of oxidative phosphorylation and the adenosine triphosphate-dependent transhydrogenase activity by a combination of membrane fractions from uncA− and uncB− mutant strains of Escherichia coli K12
Authors: Cox G. B. , Gibson F. , McCann L.
Published: August 2015

1. Membrane preparations from both uncA− and uncB− mutant strains of Escherichia coli K12, in which electron transport is uncoupled from phosphorylation, were fractionated by washing with a low-ionic-strength buffer. The fractionation gave a `5mm-Tris wash' and a `membrane residue' from each strain. This technique, applied to membranes from normal cells, separates the Mg2+,Ca2+-stimulated adenosine triphosphatase activity from the membrane-bound electron-transport chain and the non-energy-linked transhydrogenase activity. 2. Reconstitution of both oxidative phosphorylation and the ATP-dependent transhydrogenase activity was obtained by a combination of the `membrane residue' from strain AN249 (uncA−) with the `5mm-Tris wash' from strain AN283 (uncB−). 3. Valinomycin plus NH4+ inhibited oxidative phosphorylation both in membranes from a normal strain of E. coli and in the reconstituted membrane system derived from the mutant strains. 4. The electron-transport-dependent transhydrogenase activity was located in the membrane residue and was de-repressed in both the mutant strains. 5. The spatial and functional relationships between the proteins specified by the uncA and uncB genes and the transhydrogenase protein are discussed.
Metabolism of biphenyl. 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate: the meta-cleavage product from 2,3-dihydroxybiphenyl by Pseudomonas putida
Authors: Catelani Danilo , Colombi Antonio , Sorlini Claudia , Treccani Vittorio
Published: August 2015

1. 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid was isolated and identified from washed suspensions of Pseudomonas putida incubated in the presence of 2,3-dihydroxybiphenyl. 2. Benzoic acid was isolated from reaction mixtures of crude cell-free extracts incubated with 2,3-dihydroxybiphenyl. 3. The presence in the same reaction mixtures of either 4-hydroxy-2-oxovalerate or 2-hydroxypenta-2,4-dienoate was suggested by mass spectrometry. 4. The degradative pathway of biphenyl is discussed.
Cell-surface mucosubstances from trypsin disaggregation of normal and virus-transformed lines of baby-hamster kidney cells (Short Communication)
Authors: Minnikin S. Megan , Allen Adrian
Published: August 2015

Cell disaggregation by trypsin solubilizes significantly less mucosubstance from the surface of polyoma-virus-transformed baby-hamster kidney cells than from the same non-transformed cell line. The mucosubstance, which consists of both acid mucopolysaccharides and mucoproteins, also differs qualitatively in the two cell lines.
Enzyme forms produced from aspartate transcarbamoylase by digestion with trypsin
Authors: Heyde Elizabeth , Nagabhushanam A. , Venkataraman S.
Published: August 2015

1. The time-course of tryptic hydrolysis of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) was followed by activity measurements in the presence and absence of allosteric effectors, and by polyacrylamide-gel electrophoresis. 2. Two proteins with enzyme activity are formed in this way from native enzyme, and the isolation and some properties of these species are reported. The larger protein (10.6S) resembles native enzyme in that it contains regulatory subunits and is sensitive to allosteric effectors, as well as in a more detailed kinetic investigation. It appears from the time-course of tryptic digestion to be an intermediate in the formation of a catalytic subunit (5.5S) which is similar to, but not identical with, the catalytic subunit produced by mercurial treatment of the native enzyme. 3. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of the different enzyme forms demonstrates that trypsin can hydrolyse bonds in the catalytic polypeptide chains as well as completely remove the regulatory polypeptide chains. 4. Both preparations of catalytic subunit can recombine with regulatory subunit to form enzymes which resemble the native enzyme in being activated by ATP, although they do not appear to be inhibited by CTP. 5. This study is consistent with the models of the enzyme that propose that the catalytic subunits are held together in the native enzyme by three pairs of regulatory polypeptide chains.
Refolding of triose phosphate isomerase
Authors: Waley Stephen G.
Published: August 2015

The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E233, took place quite rapidly in 3m-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first-order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 100ng/ml), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was detected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations of subtilisin prevented regain of activity, but at lower concentrations refolding competed with proteolysis.
The alkylation of 2′-deoxyguanosine and of thymidine with diazoalkanes. Some observations on O-alkylation
Authors: Farmer P. B. , Foster A. B. , Jarman M. , Tisdale M. J.
Published: August 2015

The reaction in ether–methanol between 2′-deoxyguanosine and diazomethane or its ethyl or n-butyl homologue gives 1-, O6- and 7-alkyl-2′-deoxyguanosine. N2,O6-Dimethyl-2′-deoxyguanosine was also detected. The hydrolysis of the methyl and the ethyl derivatives gives the corresponding alkylguanines: the O6-alkyl-2′-deoxyguanosines were sequentially hydrolysed, first to 2-amino-6-alkoxypurines, subsequently to guanine. The mass spectra of O6-alkyl-2′-deoxyguanosines (methyl and ethyl) and of the corresponding 2-amino-6-alkoxypurines were determined. The reaction of diazomethane with thymidine afforded O4-methylthymidine, in addition to the previously detected 3-methylthymidine.
Preparation of insoluble collagen from human cartilage (Short Communication)
Authors: Steven Frank S. , Thomas Hilary
Published: August 2015

A simple technique is described for the removal of proteoglycans from human cartilage by sequential treatments with H2O2 followed by trypsin digestion. The resultant fibres contain no uronic acids and have the amino acid composition of highly purified collagen.
Mechanism and stereochemistry of the 5-aminolaevulinate synthetase reaction
Authors: Zaman Z. , Jordan P. M. , Akhtar M.
Published: August 2015

1. Two mechanisms for the biosynthesis of 5-aminolaevulinate from glycine and succinyl-CoA (3-carboxypropionyl-CoA) are considered. One of the mechanisms involves the retention of both the C-2 H atoms of glycine during the synthesis of 5-aminolaevulinate, whereas the other predicts the retention of only one of the C-2 H atoms of glycine. 2. Highly purified 5-aminolaevulinate synthetase from Rhodopseudomonas spheroides was used to show that the C-2 H atom of glycine with R configuration is specifically removed during the biosynthesis of 5-aminolaevulinate. 3. The mechanism of the condensation therefore differs from the analogous reaction of the biosynthesis of sphinganine from palmitoyl-CoA and serine, in which the C-2 H of serine is retained (Wiess, 1963).
The transporting proteins of cholecalciferol and 25-hydroxycholecalciferol in serum of chicks and other species. Partial purification and characterization of the chick proteins
Authors: Edelstein S. , Lawson D. E. M. , Kodicek E.
Published: August 2015

Chick serum contains two cholecalciferol-binding proteins, one of which binds mainly cholecalciferol (cholecalciferol-binding protein) and the other binds 25-hydroxycholecalciferol (25-hydroxycholecalciferol-binding protein). By means of Cohn fractionation, (NH4)2SO4 precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephadex and an additional gel-filtration step on Sephadex G-100, these two binding proteins were purified. Both proteins possess β-globulin mobility on analytical polyacrylamide-disc-gel electrophoresis, a sedimentation coefficient of 3.5S and approximate molecular weights of 60000 for the cholecalciferol-binding protein and 54000 for the 25-hydroxycholecalciferol-binding protein. Sera obtained from rat, pig, human and monkey were shown to contain a single binding protein that is responsible for the transport of both cholecalciferol and 25-hydroxycholecalciferol. In the toad the lipoproteins are used for the transport of these two steroids.
The sterols of the echinoderm Asterias rubens
Authors: Smith A. G. , Rubinstein I. , Goad L. J.
Published: August 2015

1. Twenty-two sterols were identified in the starfish Asterias rubens (Phylum, Echinodermata; Class, Asteroidea). 2. The major 4-demethyl sterols had a Δ7 bond and the C27 compound 5α-cholest-7-en-3β-ol predominated over other mono- and di-unsaturated sterols belonging to the C26, C27, C28 and C29 series. 3. Small amounts of cholest-5-en-3β-ol and 5α-cholestan-3β-ol were also present. 4. The minor sterols identified all contained either one or two methyl groups at C-4 and are considered to be potential biosynthetic precursors of 5α-cholest-7-en-3β-ol. 5. Three sterols possessing a 9β,19-cyclopropane ring were also isolated and were probably derived by the starfish from a dietary source.
Enzymic mechanisms involving concomitant transfer and hydrolysis reactions
Authors: Frère J.-M.
Published: August 2015

The kinetic parameters of ten different enzymic mechanisms in which bimolecular transfer reactions occur concomitantly with the hydrolysis of the donor molecule have been studied. The usefulness of these parameters for making a choice of mechanism is discussed. The analysis has been extended to the use of alternative substrates in bimolecular transfer reactions that proceed without the hydrolysis of the donor molecule.
Fluorescence and circular-dichroism studies on the Streptomyces R61 dd-carboxypeptidase–transpeptidase. Penicillin binding by the enzyme
Authors: Nieto Manuel , Perkins Harold R. , Frère Jean-Marie , Ghuysen Jean-Marie
Published: August 2015

The circular dichroism of the dd-carboxypeptidase–transpeptidase from Streptomyces R61 shows in the near u.v. a set of weak extrema at 289nm (positive) and at 282, 275 and 268nm (all negative). In the far u.v. it shows negative extrema at 217–218 and 208nm, crossover at 202nm and a positive maximum at about 194nm. The u.v. absorption of the enzyme shows it to contain tyrosine and tryptophan in approx. 3.4:1 ratio. The enzyme is fluorescent with a maximum emission at 318–320nm. The near-u.v. circular dichroism of the protein is extensively affected by binding of penicillin G, but the far u.v. is unaffected. Binding of the antibiotic also causes quenching of the fluorescence of the enzyme. The latter effect has been used to study the binding of penicillin G to the enzyme and the influence exerted upon it by salts, denaturants and peptide substrates and inhibitors. High-affinity binding of penicillin appears to be comparatively slow and reversible, and can occur under conditions in which the protein is enzymically inactive. The thermal denaturation of the enzyme in guanidinium chloride at pH7 is affected by binding of the antibiotic. The presence of even large concentrations of β-mercaptoethanol neither impaired the activity of the enzyme nor prevented its inhibition by penicillin G or cephalosporin C. A new hypothesis for the molecular mechanism of the interaction of the enzyme with penicillin is proposed.
A new general method for the assessment of the molecular-weight distribution of polydisperse preparations. Its application to an intestinal epithelial glycoprotein and two dextran samples, and comparison with a monodisperse glycoprotein
Authors: Gibbons Richard A. , Dixon Stephen N. , Pocock David H.
Published: August 2015

A specimen of intestinal glycoprotein isolated from the pig and two samples of dextran, all of which are polydisperse (that is, the preparations may be regarded as consisting of a continuous distribution of molecular weights), have been examined in the ultracentrifuge under meniscus-depletion conditions at equilibrium. They are compared with each other and with a glycoprotein from Cysticercus tenuicollis cyst fluid which is almost monodisperse. The quantity c−⅓(c=concentration) is plotted against ξ (the reduced radius); this plot is linear when the molecular-weight distribution approximates to the ‘most probable’, i.e. when Mn:Mw:Mz: M(z+1)....... is as 1:2:3:4: etc. The use of this plot, and related procedures, to evaluate qualitatively and semi-quantitatively molecular-weight distribution functions where they can be realistically approximated to Schulz distributions is discussed. The theoretical basis is given in an Appendix.
The mechanism of the reaction between glutathione and 1-menaphthyl sulphate catalysed by a glutathione S-transferase from rat liver
Authors: Gillham Brian
Published: August 2015

1. The glutathione S-transferase that catalyses the reaction of 1-menaphthyl (naphth-1-ylmethyl) sulphate with GSH was purified 76-fold from rat liver. 2. The properties of the purified enzyme were studied by gel filtration and isoelectric focusing. 3. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with an Ordered Bi Bi mechanism in which the GSH adds to the enzyme before 1-menaphthyl sulphate and the products are released in the order SO42−followed by S-(1-menaphthyl)glutathione. 4. Dead-end-inhibition studies with p-aminobenzoic acid, which has been shown to be competitive with GSH and non-competitive with 1-menaphthyl sulphate, support the suggestion that an Ordered Bi Bi mechanism is operative. 5. Values were determined for some of the dissociation and Michaelis constants for the reaction of the substrates and products with the enzyme. 6. It appears that S-(1-menaphthyl)glutathione activates the enzyme when the concentration of GSH is saturating and that of 1-menaphthyl sulphate is low (of the order of its Michaelis constant).
3-Hydroxy steroid dehydrogenase activities of cortisone reductase
Authors: Gibb William , Jeffery Jonathan
Published: August 2015

The behaviour of various C19 and C18 steroids as substrates for crystalline preparations of cortisone reductase (EC 1.1.1.53) is described. 3α(Axial,3R)-, 3α(equatorial,3R)- and 3β(axial,3S)-hydroxy steroid–NAD oxidoreductase activities are demonstrated. Four pairs of the substrates differed only in the shape of the a/b ring junction, three pairs differed only in substitution at C-10, and four pairs differed only in substitution in ring d. The shape of the substrate molecule and certain substituents (e.g. 10β-methyl, 17β-hydroxy, 16-oxo or 17-oxo) altered substrate behaviour, but steroids differing considerably in shape nevertheless acted as substrates, suggesting the possibility of a large or flexible binding site. Km values varied about 10-fold, many being approx. 140μm. Vmax. values covered a greater range (about 200-fold) and the good substrates had high Vmax. values rather than low Km values.
The electrophoretic mobility of peptides on paper at pH1.9 (Short Communication)
Authors: Bailey C. J. , Ramshaw J. A. M.
Published: August 2015

The relationship between the electrophoretic mobility, the molecular weight and the charge of peptides at pH1.9 was re-investigated. It was shown that the relationship at pH1.9 is quantitatively similar to that which holds at pH6.5.
Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of their coenzyme A esters on enzymes of fatty acid oxidation
Authors: Holland P. C. , Senior A. E. , Sherratt H. S. A.
Published: August 2015

1. Pent-4-enoyl-CoA and its metabolites penta-2,4-dienoyl-CoA and acryloyl-CoA, as well as n-pentanoyl-CoA, cyclopropanecarbonyl-CoA and cyclobutanecarbonyl-CoA, were examined as substrates or inhibitors of purified enzymes of β-oxidation in an investigation to locate the site of inhibition of fatty acid oxidation by pent-4-enoate. 2. The reactions of various acyl-CoA derivatives with l-carnitine and of various acyl-l-carnitine derivatives with CoA, catalysed by carnitine acetyltransferase, were investigated and Vmax. and Km values were determined. Pent-4-enoyl-CoA and n-pentanoyl-CoA were good substrates, whereas cyclobutanecarbonyl-CoA, cyclopropanecarbonyl-CoA and acryloyl-CoA reacted more slowly. A very slow rate with penta-2,4-dienoyl-CoA was detected. Pent-4-enoyl-l-carnitine, n-pentanoyl-l-carnitine and cyclobutanecarbonyl-l-carnitine were good substrates and cyclopropanecarbonyl-l-carnitine reacted more slowly. 3. Pent-4-enoyl-CoA and n-pentanoyl-CoA were substrates for butyryl-CoA dehydrogenase and for octanoyl-CoA dehydrogenase, and both compounds were equally effective competitive inhibitors of these enzymes with butyryl-CoA or palmitoyl-CoA respectively as substrates. Vmax., Km and Ki values were determined. 4. None of the acyl-CoA derivatives inhibited enoyl-CoA hydratase or 3-hydroxybutyryl-CoA dehydrogenase. Penta-2,4-dienoyl-CoA was a substrate for enoyl-CoA hydratase when the reaction was coupled to that catalysed by 3-hydroxybutyryl-CoA dehydrogenase. 5. In a reconstituted sequence with purified enzymes crotonoyl-CoA was largely converted into acetyl-CoA, and pent-2-enoyl-CoA into acetyl-CoA and propionyl-CoA. Penta-2,4-dienoyl-CoA was slowly converted into acetyl-CoA and acryloyl-CoA. 6. Penta-2,4-dienoyl-CoA, a unique metabolite of pent-4-enoate, was the only compound that specifically inhibited an enzyme of the β-oxidation sequence, 3-oxoacyl-CoA thiolase. The formation of penta-2,4-dienoyl-CoA could explain the strong inhibition of fatty acid oxidation in intact mitochondria by pent-4-enoate.
Ribonucleic acid synthesis in porphyric rat liver induced by 3,5-dicarbethoxy-1,4-dihydrocollidine
Authors: Nawata Hajime , Kato Kenichi
Published: August 2015

1. Administration of 3,5-dicarbethoxy-1,4-dihydrocollidine to rats caused a marked increase in the activity of δ-aminolaevulinic acid synthetase as well as a slight net increase in RNA in the livers. 2. 3,5-Dicarbethoxy-1,4-dihydrocollidine primarily stimulated the synthesis of RNA in the nucleus of the liver cell. 3. The decrease in RNA synthesis after administration of this drug resulted in a rapid decrease in the activity of this induced enzyme. This was also confirmed by treatment with actinomycin D. 4. From kinetic experiments on synthesis of RNA and δ-aminolaevulinic acid synthetase in vivo the induction mechanism of this enzyme was discussed.
Respiration-driven proton translocation in Escherichia coli (Short Communication)
Authors: Lawford Hugh G. , Haddock Bruce A.
Published: August 2015

Measurements were made of the stoicheiometry of respiration-driven proton translocation coupled to the oxidation of NAD(P)-linked or flavin-linked substrates in intact cells of Escherichia coli. Observed stoicheiometries (→H+/O quotient; Mitchell, 1966) were approx. 4 with l-malate as substrate and approx. 2 for succinate, d-lactate and glycerol oxidation. It is concluded that the potential number of equivalent energy-conservation sites associated with the respiratory chain is 2 in aerobically grown cells of E. coli harvested during the exponential phase of growth.
The role of multivalent cations in the uptake and oxidation of glucose by Pseudomonas fluorescens (Short Communication)
Authors: Walker Cynthia A. , Durham Norman N.
Published: August 2015

The effects of Mg2+, Mn2+, Ca2+ and Fe3+ on the uptake and oxidation of glucose in induced and non-induced Pseudomonas fluorescens cells were studied. Mg2+ is the most effective single metal ion in the uptake of glucose by the cell, and it functions in membrane stabilization and enzymic activity. Ca2+ will substitute for Mg2+, but Mn2+ and Fe3+ will not.
Lack of inhibition by l-1-chloro-4-phenyl-3-toluene-p-sulphoamidobutan-2-one (l-1-tosylamido-2-phenylethyl chloromethyl ketone) of the formation of bacterial polypeptide chain initiation complex (Short Communication)
Authors: Jonák J. , Clark B. F. C.
Published: August 2015

The chymotrypsin inhibitor l-1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one does not inhibit the function of the initiation factor during the formation of the polypeptide chain initiation complex in vitro. Since the inhibitor has been shown previously to inhibit polypeptide chain elongation by reacting with elongation factor EF-Tu, the inhibitor can be used to investigate the initiation and elongation steps of protein biosynthesis separately.
Relationships between concentration of hepatic intermediary metabolites and induction of the key glycolytic enzymes in vivo
Authors: Gunn J. M. , Taylor C. B.
Published: August 2015

1. The time-course for the induction of hepatic glucokinase, hexokinase, phosphofructokinase, liver-type and muscle-type pyruvate kinases in reponse to various diets and insulin has been investigated over the first 48h of change in both diabetic and non-diabetic rats. 2. The results are consistent with there being separate regulatory mechanisms for the induction of each of the three key enzymes, that is for glucokinase, phosphofructokinase and liver-type pyruvate kinase. 3. To investigate the possibility that induction of these enzymes is mediated through specific metabolites a full metabolite profile has been determined under conditions identical with those in the induction experiments and the results examined for correlations between metabolite concentrations and enzyme activities. 4. Several such relationships were detected and those between glucokinase activity and the phosphorylation state of the adenine nucleotides and between liver-type pyruvate kinase activity and the concentrations of dihydroxyacetone phosphate and pyruvate are discussed in relation to the concept of inducing metabolites. 5. It is suggested that the induction of glycolytic enzymes by insulin may be secondary to the changes in the concentration of specific hepatic metabolites brought about by the acute effects of the hormone. 6. The details of the metabolite concentrations in the various experimental states have been deposited as Supplementary Publication SUP 50021 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 can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.
The interconversion and disposal of ketone bodies in untreated and injured post-absorptive rats
Authors: Barton Roger N.
Published: August 2015

[3-14C]Acetoacetate and β-hydroxy[3-14C]butyrate were used to investigate the kinetics of ketone body metabolism in rats 3h after bilateral hind-limb ischaemia and in controls, both groups being in the post-absorptive state and in a 20°C environment. Calculations were carried out as described by Heath & Barton (1973) and the following conclusions were reached. 1. In both injured and control rats, the rates of irreversible disposal (extrahepatic utilization) of β-hydroxybutyrate and acetoacetate were proportional within experimental error to their blood concentrations up to at least 0.4mm (the maximum found in these rats), implying that they were determined, via these concentrations, by the rates of production by the liver. 2. Conversion of blood β-hydroxybutyrate into blood acetoacetate took place mainly in the liver, but the reverse process occurred mainly in extrahepatic tissues. 3. The ‘metabolic clearance rate’ (the volume of blood which, if completely cleared of substrate in unit time, would give a disposal rate equal to that in the whole animal) was calculated for β-hydroxybutyrate and acetoacetate. Comparison with the cardiac output showed that in control rats the proportion of circulating β-hydroxybutyrate extracted was lower than that of acetoacetate, clearance of which appeared almost complete. After injury both metabolic clearance rates decreased, probably because of the lower cardiac output. 4. After injury, because the average blood concentrations of ketone bodies, especially acetoacetate, were higher, the mean total rate of disposal also increased. Assuming complete oxidation, the mean contribution of ketone bodies to the whole body O2 consumption rose from 7 to 15%.
Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Requirement for J chain and a disulphide-exchanging enzyme for polymerization
Authors: Corte E. Della , Parkhouse R. M. E.
Published: August 2015

Mouse myeloma cells secreting 19S IgM (immunoglobulin M) (MOPC 104E and TEPC 183) or monomer and polymer IgA (immunoglobulin A) (MOPC 315) were incubated with radioactive leucine and the intracellular and secreted immunoglobulins and immunoglobulin subunits were prepared by preparative sucrose-density-gradient centrifugation. Samples were reduced in the presence or absence of isolated J chain, passed over Sephadex G-25 and then incubated at 37°C for 30min with or without a source of disulphide-interchange enzyme. The extent of reassembly of reduced subunits was then evaluated by electrophoresis in polyacrylamide gels. Provided that J chain and the disulphide-interchange enzyme were supplied, both IgM and IgA could be assembled from their respective subunits, obtained by reductive cleavage of polymeric forms. Under similar conditions, assembly of polymeric forms from intracellular or secreted 7S monomer subunits also occurred. Under these conditions polymerization was total, there being no residue of the monomeric form. Reassembly did not occur in the absence of either J chain or the enzyme. All of the J chain released from IgM by reductive cleavage was incorporated back into the reassembled polymer. The J chain is therefore likely to be an essential structural requirement for polymeric immunoglobulins. A variety of controls ruled out non-specific interactions, and further suggested that the amino acid sequence of polypeptide chains determines the specificity of polymerization. The fact that intracellular IgA and IgM monomer subunits known to be deficient in galactose and fucose can be completely polymerized suggests that the addition of carbohydrate does not control polymerization.
Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Control of polymerization by J chain
Authors: Parkhouse R. M. E. , Corte E. Della
Published: August 2015

Cell suspensions of mouse plasma-cell tumours secreting IgA (immunoglobulin A) and IgM (immunoglobulin M) were incubated with radioactive leucine for various periods of time. The secreted immunoglobulins were precipitated from the culture medium with specific rabbit antisera to determine the relative distribution of radioactivity among the different molecular species, and to estimate the fraction of total radioactivity in the J chain. For IgM-secreting cells there is a balanced synthesis of 7S subunits and J chains, and the secreted product is uniformly assembled to the pentamer. In cells secreting IgA, however, the results demonstrate that the pool of intracellular J chain is less than the intracellular IgA pool. The concentration of J chain is therefore limiting and is less than the requirement for complete polymerization. The major factor that determines whether an intracellular monomer is secreted as such or is polymerized with the addition of J chain is therefore the amount of intracellular J chain. When this is limiting, as it is in cells secreting IgA, then monomer will be secreted.
The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation
Authors: Stambolova M. A. , Cox D. , Mathias A. P.
Published: August 2015

1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg2+, glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [3H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [3H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).
Rapid transport of phosphatidylcholine occurring simultaneously with protein transport in the frog sciatic nerve
Authors: Abe Teruo , Haga Tatsuya , Kurokawa Masanori
Published: August 2015

1. Either l-[4,5-3H]leucine or [Me-3H]choline, or both l-[U-14C]leucine and [Me-3H]-choline, were injected into the ninth dorsal root ganglion of the frog, and peripheral transport of labelled proteins and/or phospholipids, mostly phosphatidylcholine, was studied by analysis of consecutive segments of the sciatic nerve. 2. At 25°C, approx. 5% of the3H-labelled protein was transported at the rate of 152mm/day. The rate was temperature-dependent with the Q10 value of 2.6. The flow was completely blocked by the local application of colchicine, but was unaffected by cytochalasin D. 3. [Me-3H]-Choline was incorporated into phosphatidylcholine at a comparatively slow rate, but was transported in the nerve at a rate equivalent to that for3H-labelled proteins. 4. The simultaneous transport of phosphatidylcholine and the protein was further supported in the double-labelling experiments by an identical transport rate of3H-labelled phosphatidylcholine and14C-labelled proteins, by their identical temperature dependence, by simultaneous blockade with colchicine, and also by the parallel distribution of the two labels in subcellular fractions. Specific radioactivities on a protein basis of both3H and14C labels were highest in microsomal subfractions enriched with Na+-plus-K+-stimulated adenosine triphosphatase and acetylcholinesterase. It is suggested that3H-labelled phosphatidylcholine and14C-labelled proteins transported in the nerve reside in the same structural entity, most probably a membrane component.
Preferential synthesis of a membrane-associated protein by free polyribosomes (Short Communication)
Authors: Lowe Dorrit , Hallinan T.
Published: August 2015

Isolated free polyribosomes from rat liver appear to synthesize NADPH–cytochome c reductase in vitro four times as efficiently as do membrane-bound polyribosomes. The observed synthesis by the latter could result from contamination with free polyribosomes, but an alternative explanation is also suggested.
Association of nascent polypeptide and transfer ribonucleic acid with 30S ribosomal subunits
Authors: Mirza M. Amin A. , Cannon Michael , Anderson Margaret L. M.
Published: August 2015

1. Crude extracts of Escherichia coli programmed in protein synthesis by endogenous mRNA have incorporated amino acids into protein. Analysis of such extracts by sucrose-gradient centrifugation in low Mg2+concentration has revealed that 30S ribosomal subunits carry associated radioactive material of which a considerable proportion can be removed from ribosomes by treatment of pre-labelled extracts with puromycin. 2. Gradient analyses of incorporations carried out in the additional presence of added32P-labelled tRNA have indicated that tRNA sediments in the regions of the newly synthesized nascent protein and that both labels are associated with all ribosomal components detected on the gradients under the experimental conditions employed. 3. 30S ribosomal subunits carrying both32P and14C labels have been isolated, disrupted with sodium dodecyl sulphate, and analysed by chromatography on Sephadex G-200 columns. Both labels elute closely together and well away from a tRNA marker analysed under identical conditions. 4. It is proposed that 30S ribosomal subunits, isolated from extracts which have synthesized nascent peptides under the direction of endogenous mRNA, carry associated peptidyl-tRNA.
The control of synthesis of bacterial cell walls. Interaction in the synthesis of nucleotide precursors
Authors: Anderson Raymond G. , Douglas L. Julia , Hussey Helen , Baddiley James
Published: August 2015

Phosphoenolpyruvate–UDP-N-acetylglucosamine enolpyruvyltransferase, UDP-N-acetylglucosamine pyrophosphorylase and CDP-glycerol pyrophosphorylase activities were demonstrated in soluble extracts from Bacillus licheniformis A.T.C.C. 9945. The effect of various nucleotides, sugar nucleotides and sugar phosphates on the nucleotide pyrophosphorylases was investigated. UDP-N-acetylglucosamine pyrophosphorylase was inhibited by UDP-MurAc-pentapeptide (UDP-N-acetylmuramyl-l-alanyl-d-glutamyl- meso-diaminopimelyl-d-alanyl -d-alanine) and CDP-glycerol. CDP-glycerol pyrophosphorylase was inhibited by UDP-MurAc-pentapeptide and stimulated by UDP-N-acetylglucosamine. Interaction between a precursor of one cell-wall polymer and an enzyme involved in the synthesis of a precursor of a second polymer has therefore been demonstrated. The possible role of such interaction in the control of bacterial cell-wall synthesis is discussed. Of the other compounds investigated mono- and di-nucleotides were shown to be inhibitory, indicating that nucleotide pyrophosphorylase activities may be influenced by the energy charge of the cell.
Electrokinetic properties of isolated cerebral-cortex synaptic vesicles
Authors: McLaughlin Jack , Case Kenneth R. , Bosmann H. Bruce
Published: August 2015

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of −3.55μm·s-1·V-1·cm in saline–sorbitol, pH7.2, at 25°C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pKa in the range 3.0–3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn2+or Ca2+was more effective in decreasing vesicle mobility than was Mg2+, Sr2+or Ba2+. The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.
The metabolism of benzene by bacteria. Purification and some properties of the enzyme cis-1,2-dihydroxycyclohexa-3,5-diene (nicotinamide–adenine dinucleotide) oxidoreductase (cis-benzene glycol dehydrogenase)
Authors: Axcell B. C. , Geary P. J.
Published: August 2015

1. cis-Benzene glycol dehydrogenase was purified to a homogeneous state from a species of Pseudomonas grown with benzene as the major carbon source. 2. The enzyme was specific for the cis-isomer of its substrate and required NAD+as hydrogen acceptor. 3. Partial inactivation of the enzyme, which was observed during purification, could be reversed by the addition of Fe2+and GSH. 4. A molecular weight of 440000 was calculated from data obtained by sedimentation-velocity and diffusion analysis in the ultracentrifuge. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis indicated a subunit of molecular weight 110000. 5. p-Chloromercuribenzoic acid and 1,10-phenanthroline were shown to inhibit the enzyme.
Alterations in glycosaminoglycan metabolism in β-aminopropionitrile-treated chick embryos
Authors: Elders M. Joycelyn , Smith James D. , Smith W. Grady , Hughes Edwin R.
Published: August 2015

1. Na235SO4, [1-14C]glucosamine and [1-14C]acetate were used as precursors of the sulphated glycosaminoglycans to study the biochemical effect of β-aminopropionitrile in chick embryos. The incorporation of all three precursors was decreased in the treated embryos between days 7 and 10 of embryonic development. No inhibition of incorporation of these precursors occurred between days 16 and 20 of embryonic development at the dosages of β-aminopropionitrile used. 2. β-Aminopropionitrile treatment also decreased the amount of N-acetylhexosamines in the chick embryo and decreased the percentage of the hexosamine esterified by nucleotides. Respiration was decreased by homogenates prepared from treated embryos. Likewise, UDP-xylosyl- and UDP-galactosyl-transferase activities were decreased in treated embryos and cartilage from embryos and growing chicks. 3. The data suggest that β-aminopropionitrile, in addition to the known lathyrogenic activity, either is or gives rise to a potent metabolic poison that interferes with basic cellular metabolism. The results are consistent with a decreased rate of ATP generation as an explanation for the decrease in glycosaminoglycan synthesis.
Comparison of the turnover patterns of total and individual muscle proteins in normal mice and those with hereditary muscular dystrophy
Authors: Kitchin S. E. , Watts D. C.
Published: August 2015

1. The incorporation of amino acids into hindleg muscle proteins of normal and dystrophic mice was measured ½h to 16 days after administration of the radioactive pulse. 2. Dystrophic animals showed a faster initial rate of incorporation into total and soluble proteins in the first few hours after injection, but the extent of incorporation relative to the size of the amino acid pool was similar in both. There was little difference between the overall degradation rates although this started later in the dystrophic proteins. An initial fast phase of degradation reached a plateau after 3 days whereupon the residual label in the protein remained constant up to 16 days after injection. 3. Analyses of individual radioactive proteins fractionated by polyacrylamide-gel electrophoresis showed that the distribution of label was similar in all the soluble proteins from normal and dystrophic muscle. Time-course experiments revealed that in dystrophic mice the two major soluble proteins of the muscle, creatine kinase and adenylate kinase, initially incorporated 2–3 times more label relative to the initial size of the precursor pool. This label was then lost equally rapidly and the final plateau value was much less than that in normal mice. This initial peak of activity was not observed in normal mice. 4. A group of dehydrogenases showed similar initial turnover patterns in both dystrophic and normal mice but the final plateau value was much higher in the former. 5. The results provide support for the hypothesis that there is no obvious defect in the protein synthetic machinery of dystrophic muscle. However, certain proteins do show anomalous turnover patterns relative to those in normal animals. A single structural gene mutation giving rise to one particularly unstable and readily degradable muscle protein is excluded as the cause of the dystrophy.
Intra-axonal transport and turnover of neurophysins in the rat. A proposal for a possible origin of the minor neurophysin component
Authors: Burford Graham D. , Pickering Brian T.
Published: August 2015

1. Radioactivity associated with the three neurophysins in the neural lobe of the rat was determined at intervals up to 5 weeks after an intracisternal injection of [35S]cysteine. 2. The radioactivity associated with the two major neurophysins (one supposedly associated with vasopressin and the other with oxytocin) increased linearly for 12h after the injection and the ratio of the rates of increase in the two proteins was very similar to the ratio of vasopressin to oxytocin in the gland. 3. From 12h onwards the radioactivity associated with each major neurophysin declined exponentially but the half-life of the supposed oxytocin–neurophysin (13.3 days) was shorter than that for the supposed vasopressin–neurophysin (19.8 days). 4. The kinetics of labelling of the minor neurophysin was quite different from that of the two major ones. It became slowly labelled during 3–5 days after injection and the radioactivity hardly decreased during the following 4 weeks. 5. The data could support the hypothesis that the minor neurophysin is a metabolic product of oxytocin–neurophysin. The exponential rate of disappearance of radioactivity from oxytocin–neurophysin and the minor component taken together has a rate constant similar to that for vasopressin–neurophysin (e.g. half-life=18.9 days).
An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A
Authors: Middleton B. , Tubbs P. K.
Published: August 2015

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3′-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ‘burst’ release of CoA proportional in amount to the quantity of enzyme present. The ‘burst’ of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-14C]acetyl-CoA yielded protein-bound acetyl groups. The Keq. for the acetylation was 1.2 at pH7.0 and 4°C. Acetyl-labelled synthase was isolated free from [1-14C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-14C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-14C]acetyl-CoA or 3-hydroxy-3-methyl-[14C]glutaryl-CoA respectively. 4. The isolated [1-14C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4°C with a first-order rate constant of 0.005min-1. This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.