bluuebunny/crossref_metadata_2025 huggingface dataset:
The origin and structures of dimeric fatty acids from the anaerobic reaction between soya-bean lipoxygenase, linoleic acid and its hydroperoxide
Authors: Garssen G. J. , Vliegenthart J. F. G. , Boldingh J.
Published: August 2015

In an anaerobic system soya-bean lipoxygenase catalyses in the presence of linoleic acid and l-13-hydroperoxyoctadeca-cis-9-trans-11-dienoic acid the formation of dimeric fatty acids and of carbonyl compounds. The analogous reaction does not take place when d-9-hydroperoxyoctadeca-trans-10-cis-12-dienoic acid is used instead of the 13-hydroperoxy isomer. Non-oxygenated dimers stem directly from linoleic acid and have C(11)–C(13′) or –C(9′) and C(13)–C(13′) or –C(9′) linkages. Dimers that contain oxygen originate from linoleic acid and linoleic acid hydroperoxide. It is most likely that the oxygen is present in epoxy groups.
A polymer of N-acetylglucosamine 1-phosphate in the wall of Staphylococcus lactis 2102
Authors: Archibald A. R. , Stafford G. H.
Published: August 2015

1. Walls of Staphylococcus lactis 2102 contain about 40% of a phosphorylated polysaccharide, which was isolated by extraction with cold trichloroacetic acid, with dilute NaOH, and also by digestion with a Flavobacterium peptidase. 2. The purified polymer contained equimolar proportions of N-acetylglucosamine and phosphate as its sole constituents and was readily hydrolysed under gentle acidic conditions to N-acetylglucosamine 6-phosphate. 3. Studies on the intact polymer showed that it is linear and that adjacent acetamido sugar units are joined by phosphodiester bonds between their 1- and 6-positions, the glycosidic linkages having the α-configuration. This polymer is thus the simplest of the known microbial wall polymers possessing sugar 1-phosphate linkages. 4. Alkali degradation of the extracted polymer proceeds predominantly in a stepwise manner from the reducing end, but evidence was obtained for the direct hydrolysis of some of the inter-unit phosphodiester groups.
Some examples of the use of computer-produced contour plots in the fitting of enzyme rate equations to reaction-velocity measurements
Authors: Ottaway J. H. , Apps D. K.
Published: August 2015

The use of computer-based isotach plots, relating reaction velocity to simultaneous variation of two substrates or effectors of an enzyme, in producing estimates of the parameters of enzyme rate equations was investigated. The computer program (‘SYMAP’) incorporates an interpolation algorithm, and the superiority of this over visual estimation in producing interpolated velocity values for the estimation of parameter values by conventional double-reciprocal plots is described. The usefulness of the SYMAP program in monitoring the process of fitting data obtained by simultaneous changes in two experimental variables is also described. It is shown that if the residual errors are weighted by a procedure described elsewhere (Ottaway, 1971b, 1973), the percentage error of the computed velocity is distributed evenly over a plot which contains a 100-fold variation in the concentration of one substrate and a 500-fold variation in the concentration of Mg2+, and in which the velocity of the reaction (that catalysed by NAD kinase) varies over a 60-fold range. The two-dimensional percentage error plot was used to assess the limits within which an incomplete inhibition equation is valid, and to detect a discrepancy in an expected good fit, caused by an impurity in one of the substrates.
Shapes of curves of pH-dependence of reactions
Authors: Dixon Henry B. F.
Published: August 2015

A simple case is considered in which the rate of a two-step reaction depends on pH because the intermediate formed in the first step has to gain (or lose) a proton before it can react in the second step, and in which the rate-determining step therefore changes with pH. The curves of reaction rate against pH are shown to be symmetrical, and the sharpest peak possible has a width at half its height of 1.53pH units, i.e. of 2log(3+2√2). Any particular curve for this situation proves to be identical with a curve that could be generated for the pH-dependence of a single-step reaction in which the rate is proportional to the concentration of a particular ionic form of a reactant. Curves for the latter situation, however, can have forms impossible for the former case in which the rate-determining step changes, but only if the protonations that activate and deactivate the reactant are co-operative. The peak can then become even sharper, and its width at half its height can fall to 1.14pH units, i.e. to 2log(2+√3).
The purification, composition and specificity of wheat-germ agglutinin
Authors: Allen A. K. , Neuberger A. , Sharon N.
Published: August 2015

1. The purification of wheat-germ agglutinin from commercial wheat germ is described. By ion-exchange chromatography three active proteins (isolectins) were separated, one of which was examined in detail. 2. The amino acid composition is unusual, as 20% of residues are half-cystine and 21% are glycine. Unlike most lectins and contrary to previous reports, this protein is not a glycoprotein. 3. The efficiency of various saccharides as inhibitors of the agglutination reaction was investigated and from this the specificity of the binding site was inferred. Of monosaccharides, only derivatives of glucose with a 2-acetamido group and a free 3-hydroxyl group are effective inhibitors, and glycosides of either anomeric configuration are bound. Oligosaccharides are much more powerful inhibitors of agglutination than are monosaccharides. 4. It is proposed that the binding site consists of three or four subsites with differing specificities, in a cleft in the molecule resembling that proposed for hen's-egg-white lysozyme.
Effect of alcohols and neutral salt on the thermal stability of soluble and precipitated acid-soluble collagen
Authors: Russell A. E.
Published: August 2015

The effects of mono- and poly-hydric alcohols in the presence of KCl on the intrinsic stability of collagen molecules in dilute acid solution were compared with corresponding solvent and salt effects on the increased stability of the aggregated molecules in salt-precipitated fibrils. Salt addition decreased solubility and increased the thermal stability of fibrils, but progressively decreased the stability of collagen molecules in solution. In contrast, the alcohols enhanced solubility and decreased fibril stability, the effects increasing with solvent hydrocarbon chain length and with decreasing hydroxyl/methylene-group ratio. Molar destabilization of dissolved collagen by alcohols was lower than for fibrils, and at low salt concentration, both ethylene glycol and glycerol were structural stabilizers. Electron-micrograph studies indicated that salt-precipitated fibrils tended to adopt the native aggregation mode, and qualitatively similar solvent effects were observed in insoluble collagens. Implications of the experimental findings are discussed in terms of a model in which electrostatic and apolar interactions mainly govern the excess of stability in collagen fibrils whereas intrinsic stability of single molecules is a function of polar interactions and polypeptide-chain rigidity.
Synthesis of deoxyribonucleic acid in BHK-21/C13 cells
Authors: Hayton Geoffrey J. , Pearson Colin K. , Scaife Jeremy R. , Keir Hamish M.
Published: August 2015

Newly synthesized 3H-labelled DNA was extracted from baby hamster kidney cells (BHK-21/C13 cells) and was shown to possess single-stranded properties when examined by column chromatography on benzoylated naphthoylated DEAE-cellulose, hydroxyapatite and methylated albumin on kieselguhr, and by its affinity for nitrocellulose filters. Some of the newly synthesized DNA was shown to be of lower molecular weight than the bulk of the DNA when examined by alkaline sucrose-density-gradient centrifugation. The properties observed were not affected by treatment of the DNA with ribonuclease, Pronase or amylase. The effect of the size of the DNA on its observed properties was examined and is discussed. It is concluded that DNA synthesis in BHK-21/C13 cells proceeds according to the discontinuous-mechanism model in at least one of the strands.
The characteristics of the ‘peroxidatic’ reaction of catalase in ethanol oxidation
Authors: Oshino Nozomu , Oshino Reiko , Chance Britton
Published: August 2015

Ethanol oxidation by rat liver catalase (the ‘peroxidatic’ reaction) was studied quantitatively with respect to the rate of H2O2 generation, catalase haem concentration, ethanol concentration and the steady-state concentration of the catalase–H2O2 intermediate (Compound I). At a low ratio of H2O2-generation rate to catalase haem concentration, the rate of ethanol oxidation was independent of the catalase haem concentration. The magnitude of the inhibition of ethanol oxidation by cyanide was not paralleled by the formation of the catalase–cyanide complex and was altered greatly by varying either the ethanol concentration or the ratio of the rate of H2O2 generation to catalase haem concentration. The ethanol concentration producing a half-maximal activity was also dependent on the ratio of the H2O2-generation rate to catalase haem concentration. These phenomena are explained by changes in the proportion of the ‘catalatic’ and ‘peroxidatic’ reactions in the overall H2O2-decomposition reaction. There was a correlation between the proportion of the ‘peroxidatic’ reaction in the overall catalase reaction and the steady-state concentration of the catalase–H2O2 intermediate. Regardless of the concentration of ethanol and the rate of H2O2 generation, a half-saturation of the steady state of the catalase–H2O2 intermediate indicated that about 45% of the H2O2 was being utilized by the ethanol-oxidation reaction. The results reported show that the experimental results in the study on the ‘microsomal ethanol-oxidation system’ may be reinterpreted and the catalase ‘peroxidatic’ reaction provides a quantitative explanation for the activity hitherto attributed to the ‘microsomal ethanol-oxidation system’.
Nucleotide sequences of similar size from the coliphage R17 genome
Authors: Rensing Ulrich F. E. , Coulson Alan , Thompson E. O. P.
Published: August 2015

A sequence of 33 nucleotides from the coliphage R17 RNA genome was determined. It constitutes the main component of a mixture of fragments that migrate together on electrophoresis in a separation according to molecular weight. Fragments of comparable chain length from 3′ end of RNA from coliphage R17, from a region preceding and overlapping the coat-protein cistron ribosome binding site and from the beginning of the A-protein cistron, were also found and characterized. ‘Hairpin’-like secondary structures are proposed for the longer fragments, one of which appears to have a tetranucleotide excised in the loop region.
The reaction of protein amino groups with methyl 5-iodopyridine-2-carboximidate. A possible general method of preparing isomorphous heavy-atom derivatives of proteins
Authors: Riley Malcolm , Perham Richard N.
Published: August 2015

1. The synthesis of methyl 5-iodopyridine-2-carboximidate and its reaction with amino groups of model compounds and performic acid-oxidized insulin are described. The reagent was designed to introduce heavy atoms into specific sites in proteins. 2. Specific reaction with the amino groups of oxidized insulin can be achieved under reasonably mild conditions giving rise to the corresponding N-monosubstituted amidines. 3. The extent of reaction of this reagent with protein amino groups can be readily determined by difference spectroscopy. Modification of lysine residues inhibits tryptic cleavage at such residues, and this can be of assistance in establishing the site of modification in the primary structure. 4. Evidence is presented to show that methyl 5-iodopyridine-2-carboximidate can react specifically, at pH5.0, with the aromatic amino group of 3-amino-l-tyrosine; the final product of this reaction is a 2-arylbenzoxazole. 5. The use of this reagent as a general method for preparing heavy-atom isomorphous derivatives of proteins is discussed.
Investigations of the structural function of the J chain in human immunoglobulin M
Authors: Ricardo Manuel J. , Inman Franklin P.
Published: August 2015

Human IgM molecules were treated with Na2SO3 or mercaptoethylamine in concentrations ranging from 2 to 14mm or 2 to 22mm respectively. The dissociation of IgM to IgMs varied from 0% to 100%. At the intermediate concentrations of either reagent the amount of freed J chains was less than expected. In an attempt to find an explanation for this, IgM was partially dissociated to IgMs with mercaptoethylamine. The IgMs isolated by gel filtration was divided according to the ascending and descending portions of the elution curve. These portions were treated with 24mm-mercaptoethylamine and analysed for the presence of J chains. Only the ascending portion contained free J chains. Thus, after mild reduction where not all the IgM molecules are dissociated to IgMs, some J chains remain covalently attached to some IgMs molecules although most of the J chains are freed. It was concluded that the J chain could serve as a ‘hitch’ for IgMs molecules forming intact IgM.
Streptomyces
dd-carboxypeptidase as transpeptidases. The specificity for amino compounds acting as carboxyl acceptors
Authors: Perkins Harold R. , Nieto Manuel , Frère Jean-Marie , Leyh-Bouille Mélina , Ghuysen Jean-Marie
Published: August 2015

The ability of the water-soluble dd-carboxypeptidases of Streptomyces strains albus G, R61, K11 and R39 to perform transpeptidation was studied. The donor was diacetyl-l-lysyl-d-alanyl-d-alanine, and a whole range of amino acids, peptides and structurally related amino compounds were tested for acceptor function. No compound tested was an acceptor for the enzyme from strain albus G whereas the enzymes from strains R61 and K11 could utilize with varying efficiency a wide range of substances including peptides with N-terminal glycine or d-alanine, ω-amino acids, aminohexuronic acids, 6-aminopenicillanic acid and d-cycloserine. Certain peptides, when present in higher concentration, inhibited the transpeptidation observed at lower concentration. The enzyme from strain R39 would not use any dipeptide as an acceptor, but a few compounds that were not glycine or α-amino acids of the d-configuration did function thus. These were d-cycloserine and the lactams of meso- or racemic-diaminoadipic acid.
Studies on the coagulant enzyme from Agkistrodon rhodostoma venom. Isolation and some properties of the enzyme
Authors: Hatton M. W. C.
Published: August 2015

1. Arvin, a commercial preparation of the coagulant activity from the venom of Agkistrodon rhodostoma, is shown to contain a non-coagulant caseinolytic fraction. 2. A method is described for the purification of the coagulant enzyme free from any detectable contaminating protein. 3. The coagulant enzyme is identified as a glycoprotein which probably consists of a single polypeptide chain containing approx. 29% by weight of carbohydrate. Amino acid and carbohydrate analyses are reported and the N- and C-terminal amino acid residues identified. 4. Electrophoresis on polyacrylamide gel reveals the polymorphic nature of the glycoprotein. Five forms of the enzyme are observed. 5. The coagulant action is correlated with an arginine esterase activity and kinetic properties are studied with both arginine and lysine esters as substrates. The inhibitory nature of guanidine and arginine toward the esterase activity is reported.
Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)
Authors: Stribling Donald , Perham Richard N.
Published: August 2015

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.
The effect of glucose, insulin and noradrenaline on lipolysis and on the concentrations of adenosine 3′:5′-cyclic monophosphate and adenosine 5′-triphosphate in adipose tissue
Authors: Knight Brian L. , Iliffe Jill
Published: August 2015

Glycerol release and tissue concentrations of ATP and cyclic AMP were followed during the incubation of adipose tissue with or without glucose, insulin and noradrenaline. Glucose plus insulin or, to a lesser extent, glucose alone increased the accumulation of glycerol during incubations both with and without noradrenaline by slowing the decline in the rate of glycerol release with time. Insulin alone decreased the accumulation by accelerating the fall in glycerol release. In the absence of noradrenaline, ATP and cyclic AMP concentrations were not significantly affected by insulin or glucose. With noradrenaline or noradrenaline plus insulin the ATP concentration gradually fell. With noradrenaline plus glucose the ATP concentration fell rapidly and then stabilized, or, if insulin was also present, returned to the control value. In the presence of noradrenaline, the concentration of cyclic AMP rose during the first 20min and then fell. Insulin lowered the peak concentration of cyclic AMP, but glucose had no effect either on the peak value or the fall in the concentration of the nucleotide. The increase and fall in the concentration of cyclic AMP with noradrenaline or noradrenaline plus insulin bore similarities to the increase and decline in the lipolytic rate in incubations without glucose. It is proposed that glucose stimulates ATP production by furnishing glycerol 1-phosphate and thus removing free fatty acids, but that it can influence lipolysis by a mechanism which is distinct from any which is mediated by free fatty acids, possibly by inhibiting the inactivation of the lipase.
Control of ovarian cholesterol ester biosynthesis
Authors: Flint A. P. F. , Grinwich D. L. , Armstrong D. T.
Published: August 2015

1. Experimental evidence is presented for a role of progesterone and 20α-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [14C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3′:5′-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [14C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20α-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20α-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20α-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.
Amino acid metabolism of experimental granulation tissue in vitro
Authors: Aalto Maija , Lampiaho Kirsti , Pikkarainen J. , Kulonen E.
Published: August 2015

1. The intracellular volume in granulation tissue was about 15% of the total urea space. 2. The experimental granuloma has a greater ability to retain amino acids during the proliferation phase than later during the synthesis of collagen. 3. The synthesis of collagen and other proteins by granulation tissue is related to the concentrations of proline and glutamic acid in the medium. 4. The rate of synthesis of proline from glutamic acid in granulation-tissue slices is greatest during collagen synthesis. It is enhanced by lactate. 5. Extracellular cations influence the synthesis of collagen and ouabain is inhibitory. Synthesis of other proteins is less sensitive in this respect. 6. It is suggested that the synthesis of collagen is related to the supply of certain amino acids, especially proline, and hence to the redox balance, and also to the function of the cell wall.
Serum factors affecting the incorporation of [3H]uridine by lymphocytes stimulated by concanavalin A. Studies of the role of complement
Authors: Milthorp P. , Forsdyke D. R.
Published: August 2015

1. Rat lymph-node cells cultured in serum and medium 199 were activated to transform and proliferate by concanavalin A. Initial cell activation was assessed by measuring the enhanced radioactive labelling of cells with [3H]uridine produced by concanavalin A during the first 6h of culture. 2. In medium containing serum the degree of activation was dependent on the ratio of concanavalin A to non-diffusible serum macromolecules; however, cells could be activated to a normal extent in a medium containing only diffusible molecules. This indicates that certain serum macromolecules buffer cell receptor sites against reaction with concanavalin A. 3. At high concanavalin A concentrations labelling was depressed below that of control cultures without concanavalin A. This inhibition of labelling (i) occurred in calf serum, but not in homologous serum, (ii) was removed by pretreatment of serum with various complement inhibitors, and (iii) first appeared after 1h of culture following an initial phase of cell activation by concanavalin A. Cells pre-labelled with [3H]uridine slowly released the label into the culture medium; this rate of release was suddenly accelerated after 1h of culture with concanavalin A if complement was present. The results suggest that inhibition of labelling requires the sequential binding of concanavalin A and then complement to the cell surface. 4. Results from experiments in which calf serum was mixed in various proportions with calf serum which had been preheated to inactivate complement, suggest (i) a requirement for complement in stoicheiometric quantities dependent on the number of cells being inhibited, and (ii) that preheated serum can inactivate complement in unheated serum. 5. The proliferative response over 3 days of culture was assessed by measuring the enhanced labelling of cells with [3H]thymidine produced by concanavalin A. In preheated calf serum two types of inhibition were noted. (i) A progressive inhibition at high concanavalin A concentrations so that the optimum response was shifted to lower concanavalin A concentrations as the duration of culture was extended; it is suggested that this reflects the secretion of complement by cultured cells. (ii) An inhibition of the optimum response appearing late in the culture period at high cell concentrations; it is suggested that this is due to the exhaustion of medium nutrients in most actively growing cultures.
d-3-Hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetic mechanism from steady-state kinetics of the reaction catalysed by the enzyme in solution and covalently attached to diethylaminoethylcellulose
Authors: Preuveneers M. J. , Peacock D. , Crook E. M. , Clark J. B. , Brocklehurst K.
Published: August 2015

1. The reversible NAD+-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0°C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate–NAD+ oxidoreductase, EC 1.1.1.30), was studied by initial-velocity, dead-end inhibition and product-inhibition analysis. 2. The reactions were carried out on (a) the soluble enzyme from Rhodopseudomonas spheroides and (b) an insoluble derivative of this enzyme prepared by its covalent attachment to DEAE-cellulose by using 2-amino-4,6-dichloro-s-triazine as coupling agent. 3. The insolubilized enzyme preparation contained 5mg of protein/g wet wt. of total material, and when freshly prepared its specific activity was 1.2μmol/min per mg of protein, which is 67% of that of the soluble dialysed enzyme. 4. The reactions catalysed by both the enzyme in solution and the insolubilized enzyme were shown to follow sequential pathways in which the nicotinamide nucleotides bind obligatorily first to the enzyme. Evidence is presented for kinetically significant ternary complexes and that the rate-limiting step(s) of both catalyses probably involves isomerization of the enzyme–nicotinamide nucleotide complexes and/or dissociation of the nicotinamide nucleotides from the enzyme. Both catalyses therefore are probably best described as ordered Bi Bi mechanisms, possibly with multiple enzyme–nicotinamide nucleotide complexes. 5. The kinetic parameters and the calculable rate constants for the catalysis by the soluble enzyme are similar to the corresponding parameters and rate constants for the catalysis by the insolubilized enzyme.
The effect of modifying lysine-126 on the physical, catalytic and regulatory properties of bovine liver glutamate dehydrogenase
Authors: Wallis Robert B. , Holbrook J. John
Published: August 2015

1. The reaction of 4-iodoacetamidosalicylate with bovine liver glutamate dehydrogenase is dependent on pH. The pH–activity curve is bell-shaped and can be described by apparent pK values of 7.8±0.2 and 9.1±0.2. 2. Enzyme in which lysine-126 has been modified by 4-iodoacetamidosalicylate has unaltered sedimentation characteristics except when measured in the presence of GTP and NADH. 3. GTP binding to the inhibited enzyme is unaltered. However, GTP can no longer promote the binding of a second molecule of NADH, since this is already bound to the inhibited enzyme without GTP. 4. The equilibrium binding of ADP, GTP, NAD–sulphite and NADH (when measured at low concentrations) was largely unchanged by modification. 5. The number of binding sites for 2-oxoglutarate to the enzyme–NADH complex were decreased by 60% in an enzyme that has been inhibited by 70%.
The purification of nicotinamide nucleotide-dependent dehydrogenases on immobilized cofactors
Authors: Lowe C. R. , Harvey M. J. , Craven D. B. , Kerfoot M. A. , Hollows M. E. , Dean P. D. G.
Published: August 2015

1. The general applicability of affinity chromatography to the purification of nicotinamide nucleotide-dependent dehydrogenases on immobilized cofactors is illustrated with several examples taken from crude systems. 2. Methods for overcoming the inevitable loss of selectivity experienced with these polymers are suggested. Effective use of the appropriate nucleotide, the second substrate and other interacting ligands can be made to selectively alter the chromatographic behaviour of the desired enzyme.
Affinity chromatography of lactate dehydrogenase on immobilized nucleotides
Authors: Lowe C. R. , Dean P. D. G.
Published: August 2015

The interaction of two isoenzymes of lactate dehydrogenase from pig heart muscle (H4) and rabbit skeletal muscle (M4), with immobilized nucleotides was examined: the effects of pH and temperature on the binding of lactate dehydrogenase were studied with immobilized NAD+ matrices. The influence of substrate, product and sulphite on the binding of heart muscle lactate dehydrogenase to immobilized NAD+ was investigated. The interaction of both lactate dehydrogenase isoenzymes with immobilized pyridine and adenine nucleotides and their derivatives were measured. The effects of these parameters on the interaction of lactate dehydrogenase with immobilized nucleotides were correlated with the known kinetic and molecular properties of the enzymes in free solution.
Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae
Authors: Hawes Catherine S. , Nicholas D. J. D.
Published: August 2015

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15°C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP2- complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the Km of each substrate was determined at infinite concentration of the other (Km MgATP2-, 0.07mm; MoO42-, 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5′-[35S]sulphatophosphate and SO42-, 35SO42- and adenosine 5′-sulphatophosphate, occurred only in the presence of either MgATP2- or PPi. This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO32-, S2O32- (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO42- was produced by SO42- (Ki=2.0mm) and non-competitive inhibition by sulphide (Ki=3.4mm). 8. Adenosine 5′-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity–substrate curves with both MgATP2- and MoO42- to sigmoidal forms.
The properties of subunits of avidin coupled to Sepharose
Authors: Green N. Michael , Toms E. John
Published: August 2015

Avidin that had been coupled to Sepharose 4B activated with CNBr retained over 90% of its biotin-binding capacity. When low concentrations of CNBr were used about 75% of the protein could be removed from the Sepharose by washing with guanidinium chloride (6 m). The remaining 25%, the covalently bound subunits, had an almost undiminished capacity for biotin but a decreased affinity. Addition of avidin subunits in guanidinium chloride to the coupled subunits followed by dilution or dialysis restored the original biotin-binding capacity and affinity. Three classes of binding sites were present in preparations of the subunits. About 25% were weak (K=5X10−8m), about one third exchanged their biotin in a few minutes (K∼10−10m) and the remainder were indistinguishable from the native tetramer. The last-named exchanged their bound biotin at a similar rate at pH5 and at pH2, they did not lose their biotin in 6 m-guanidinium chloride and they were resistant to tryptic digestion in the absence of biotin. The proportion of these stable sites could be increased to 65% when the subunits coupled to Sepharose were incubated at 37°C. This increase was reversed by guanidinium chloride, which suggested that it was caused by a temperature-dependent association of covalently linked subunits. This in turn implies a temperature-dependent mobility of the agarose matrix of the Sepharose. Analysis of the spatial distribution of subunits within the Sepharose beads led to the conclusion that the association of subunits implied that they could move through distances greater than 20nm (several hundred Å). This mobility and consequent formation of tetramer was greatly decreased when avidin subunits were coupled to Sepharose that had been cross-linked with divinyl sulphone.
The interaction of α2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism
Authors: Barrett Alan J. , Starkey Phyllis M.
Published: August 2015

1. α2-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to believe that α2-macroglobulin can bind essentially all proteinases. 2. Radiochemically labelled trypsin, chymotrypsin, cathepsin B1 and papain are bound by α2-macroglobulin in an approximately equimolar ratio. Equimolar binding was confirmed for trypsin by activesite titration. 3. Pretreatment of α2-macroglobulin with a saturating amount of one proteinase prevented the subsequent binding of another. We conclude that each molecule of α2-macroglobulin is able to react with one molecule of proteinase only. 4. α2-Macroglobulin did not react with exopeptidases, non-proteolytic hydrolases or inactive forms of endopeptidases. 5. The literature on binding and inhibition of proteinases by α2-macroglobulin is reviewed, and from consideration of this and our own work several general characteristics of the interaction can be discerned. 6. A model is proposed for the molecular mechanism of the interaction of α2-macroglobulin with proteinases. It is suggested that the enzyme cleaves a peptide bond in a sensitive region of the macroglobulin, and that this results in a conformational change in the α2-macroglobulin molecule that traps the enzyme irreversibly. Access of substrates to the active site of the enzyme becomes sterically hindered, causing inhibition that is most pronounced with large substrate molecules. 7. The possible physiological importance of the unique binding characteristics of α2-macroglobulin is discussed.
Loss of tryptophan associated with photopolymerization and yellowing of proteins exposed to light over 300nm
Authors: Dilley Keith J.
Published: August 2015

S-Carboxymethyl-lysozyme and S-carboxymethyl-ribonuclease A were irradiated with light of wavelength greater than 300nm. Photo-oxidative loss of tryptophan from the S-carboxymethyl-lysozyme was accompanied by yellowing of the protein and the formation of covalently cross-linked polymers. S-Carboxymethyl-ribonuclease, which contains no tryptophan, showed little yellowing and no polymer formation. The irradiated S-carboxymethyl-lysozyme was similar to the proteins of the brown cataractous human lens nucleus and to bovine lens proteins exposed to sunlight in vitro in that it (1) was insoluble in non-denaturing solvents, (2) contained a new fluorescence, (3) was brown in colour, and (4) contained covalent cross-links that are not disulphide bonds.
Amino acid absorption by mouse ascites-tumour cells depleted of both endogenous amino acids and adenosine triphosphate
Authors: Morville M. , Reid M. , Eddy A. A.
Published: August 2015

1. Despite the depletion of both their content of exchangeable endogenous amino acids and reserves of ATP, starved hypo-osmotically shocked preparations of the tumour cells accumulated relatively large amounts of 14C-labelled 2-aminoisobutyrate, l-alanine, glycine, l-leucine, l-methionine, l-phenylalanine and l-serine, against their respective concentration gradients, by a process apparently driven by the spontaneous flow of Na+ ions into the cellular phase. Dependent on (a) which compound was used, (b) its concentration and (c) the direction of the Na+ ion gradient, the peak value of the ratio of the cellular to extracellular amino acid concentration varied from about 0.4 to 7. 2. The extent to which ATP increased the ratio was defined for l-methionine. 3. Chemical analysis of the cellular amino acid content showed that this increased in parallel with the absorption of 14C. 4. The accumulation of l-methionine and of glycine, against their own concentration gradients, continued in the presence of either 0.3mm-ouabain or 10μg of oligomycin/ml. Thus the sodium pump was probably not involved in the process when ATP was lacking. 5. l-Leucine caused 0.72±0.12 (s.e.m.; 6) extra equivalents of Na+ to enter the shocked starved tumour cells in parallel with the uptake of leucine itself. Only a small loss of K+ was induced. 6. The influx and efflux of l-methionine in preparations depleted of ATP were both markedly accelerated by the presence of Na+ ions. 7. The observations provide further examples of the application of the ion-gradient hypothesis, according to which Na+ ions act as co-substrates of the amino acid pump. The quantitative importance of parallel Na+-independent systems was studied with a new mathematical model.
Heterogeneous distribution of glucose 6-phosphatase in rat liver microsomal fractions as shown by adaptation of a cytochemical technique
Authors: Lewis John A. , Tata Jamshed R.
Published: August 2015

1. A novel technique for the subfractionation of rat liver smooth and rough microsomal fractions according to their content of glucose 6-phosphatase is described. This technique, based on the Gomori lead histochemical procedure, involves incubation of smooth and rough microsomal fractions with low concentrations of Pb(NO3)2 and glucose 6-phosphate. Control experiments, in which enzyme was assayed in the presence of various amounts of Pb(NO3)2 or in which microsomal fractions were reisolated after incubation with low concentrations of Pb(NO3)2 and glucose 6-phosphate, showed that lead does not interfere with glucose 6-phosphatase activity. 2. Discontinuous sucrose-density-gradient centrifugation of microsomal fractions which had previously been incubated with various amounts of Pb(NO3)2 and glucose 6-phosphate showed that it is possible to subfractionate both smooth- and rough-microsomal fractions into several bands, owing to a differential modification of the density of the microsomal vesicles by the trapping of lead phosphate within them. 3. When the material in the bands obtained by density-gradient centrifugation of incubated microsomal fractions was assayed for glucose 6-phosphatase activity, it was found that the modification of the density of the microsomal fractions was directly related to their relative enrichment in glucose 6-phosphatase activity. Control experiments, in which microsomal fractions were incubated with Pb(NO3)2 and glucose 6-phosphate and then treated with EDTA, showed that the subfractionation was not due to aggregation of microsomal vesicles, lead and glucose 6-phosphate. Thus the resolution of microsomal preparations into subfractions with different glucose 6-phosphatase activities is interpreted as indicating heterogeneity of glucose 6-phosphatase distribution in the microsomal vesicles. 4. Electron micrographs of both smooth- and rough-microsomal subfractions show deposits of lead phosphate within the microsomal vesicles. The frequency and extent of these deposits correlate with the different amounts of glucose 6-phosphatase activity measured biochemically. 5. The nature of the heterogeneous distribution of glucose 6-phosphatase is discussed and the more general applicability of the technique for studying membrane fractions containing a heterogeneous distribution of phosphatases is indicated.
Studies on the biosynthesis of cytochrome c
Authors: Colleran Emer M. , Jones O. T. G.
Published: August 2015

A soluble cytochrome was isolated and purified from the slime mould Physarum polycephalum and identified as cytochrome c by room-temperature and low-temperature (77°K) difference spectroscopy. A close similarity between P. polycephalum and mammalian cytochromes c was suggested by a comparison of the initial rates of oxidation of both proteins by mammalian mitochondria. This similarity was further emphasized by redox titrations and gel-electrophoretic studies which indicated that P. polycephalum cytochrome c has an oxidation–reduction midpoint potential of +257mV at pH7.0 and a molecular weight of 12500±1500 (mean±maximum deviation for a set of six measurements). P. polycephalum exhibits an absolute requirement for protohaemin for growth. The 59Fe-labelled haemin was prepared by chemical synthesis from protoporphyrin. The purified product had a specific radioactivity of 0.8±0.02μCi/mol. Growth of P. polycephalum in the presence of [59Fe]haemin resulted in the incorporation of 59Fe into the plasmodial cytochrome c. The specific radioactivity of the cytochrome c haem was 0.36±0.02μCi/mol. The high specific radioactivity of the cytochrome haem indicates that synthesis of the holoenzyme must proceed by direct attachment of haem to the apoprotein rather than by the intermediate formation of a protoporphyrinogen–apoprotein complex. The observed decrease in the specific radioactivity of the haem group is attributed to exchange of the 59Fe with unlabelled iron in the plasmodia either before or during attachment of the haem group to the apoprotein.
The detection of the messenger ribonucleic acid for the α-lactalbumin of guinea-pig milk (Short Communication)
Authors: Campbell P. N. , McIlreavy Diana , Tarin D.
Published: August 2015

RNA was extracted from the polyribosomes isolated from the mammary glands of a lactating guinea pig and injected into Xenopus oocytes. On incubation the oocytes effected the biosynthesis of α-lactalbumin.
Amino acid production in isolated rat liver mitochondria
Authors: Ferdinand W. , Bartley W. , Broomhead V.
Published: August 2015

Amino acid analyses of mitochondrial membranes are compared with the amino acid composition of whole mitochondria (Alberti, 1964) and found to be very similar except in the cystine content. The composition of the endogenous amino acids found in freshly prepared mitochondria has been established and shown to differ considerably from the amino acid composition of membranes or whole mitochondria. The amino acids produced during anaerobic incubation of mitochondria at pH7.4, on the other hand, resemble the membrane in composition, supporting the view that neutral proteinase activity is responsible for their appearance. Aerobic incubation produces a similar pattern of amino acids except that amino acids such as proline, serine, asparagine, glutamic acid and glutamine, which can be metabolically utilized under aerobic conditions, are present to a smaller extent. The presence of large relative concentrations of endogenous taurine, cysteic acid and oxidized glutathione and the accumulation of taurine during incubation is found. The selective retention of taurine and cysteic acid within the mitochondria is established. It is proposed that the first step in the degeneration of isolated mitochondria results from lipid hydroperoxide accumulation caused by the lack of glutathione reductase in isolated mitochondria.
Biosynthesis of glycosaminoglycans by cultured mastocytoma cells
Authors: Lewis Richard G. , Spencer Alice F. , Silbert Jeremiah E.
Published: August 2015

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [35S]sulphate (and in some cases [6-3H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [3H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.
Exogenous cholesterol transport in rabbit plasma lipoproteins
Authors: Rudel L. L. , Felts J. M. , Morris M. D.
Published: August 2015

1. The appearance of exogenous cholesterol in free cholesterol and ester cholesterol of plasma chylomicra, very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins was studied in unanaesthetized rabbits after ingestion of a meal containing 5% fat and 0.08% [3H]cholesterol. 2. The specific radioactivity of ester cholesterol of VLD lipoproteins reached the highest value of any lipoprotein fraction and for each lipoprotein it increased at a faster rate and reached a higher maximum than that of free cholesterol; the maximum in VLD lipoproteins occurred later than in chylomicra. 3. The pattern of appearance of exogenous cholesterol in chylomicra and VLD lipoproteins of plasma was similar to the pattern previously observed in lymph. The specific radioactivity of ester cholesterol in plasma VLD lipoproteins was higher than that in chylomicra in spite of a larger pool size and dilution of cholesteryl esters from VLD lipoproteins produced by the liver. These results support the concept that during absorption the major portion of exogenous cholesterol is transported in VLD lipoproteins as ester cholesterol. 4. The specific radioactivity of ester cholesterol of chylomicra and VLD lipoproteins increased at a faster rate than that of LD and HD lipoproteins. However, the rate of increase and the absolute values of the specific radioactivity in LD and HD lipoproteins were identical. Since cholesteryl esters are thought not to exchange between lipoproteins, this observation supports the hypothesis that a result of VLD lipoprotein and chylomicron metabolism is the formation of LD and HD lipoproteins. 5. Results in vivo showed that the free cholesterol of individual plasma lipoproteins does not equilibrate within a period of 24h.
Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain
Authors: Deery D. J. , Taylor K. W.
Published: August 2015

1. The effects of azaserine and nicotinamide, agents which inhibit and stimulate hepatic NAD synthesis respectively, on the content of NAD+ and NADH in isolated rat islets of Langerhans incubated in vitro were studied. The effects of these compounds on the rates of insulin release, from isolated islets incubated in vitro, in response to various secretagogues were also measured. 2. Preincubation of islets in the presence of azaserine (0.3mm) caused a marked depletion of the normal islet-cell content of both NAD+ and NADH and prevented the secretion of insulin in response to stimulatory concentrations of d-glucose, xylitol, d-xylulose, l-arginine hydrochloride and l-leucine. 3. Preincubation of islets in the presence of nicotinamide (2mm) increased the islet content of NAD+ and enhanced the rate of release of insulin in response to d-glucose. Also when nicotinamide was present the inhibitory effect of azaserine on insulin release and the azaserine-induced depletion of the islet content of NAD+ and NADH was prevented. 4. Preincubation with azaserine was without effect on the stimulation of insulin release caused by theophylline or dibutyryl cyclic AMP. 5. It is suggested that insulin release caused by sugars and amino acids is dependent on the maintenance of NAD concentrations, though this may not be the case for release due to theophylline and dibutyryl cyclic AMP.