bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbial metabolism of the pyridine ring. The metabolism of pyridine-3,4-diol (3,4-dihydroxypyridine) by Agrobacterium sp
Authors: Watson G. Keith , Houghton Charles , Cain Ronald B.
Published: August 2015

1. Pyridine-3,4-diol (3,4-dihydroxypyridine, 3-hydroxypyrid-4-one), an intermediate in 4-hydroxypyridine metabolism by an Agrobacterium sp (N.C.I.B. 10413), was converted by extracts into 1mol of pyruvate, 2mol of formate and 1mol of NH3 at pH7.0. 2. Formate, but not the alternative likely product formamide, was further oxidized fivefold faster by 4-hydroxypyridine-grown washed cells than by similar organisms grown on succinate. 3. The oxidation of pyridine-3,4-diol by crude extracts at pH8.5 required 1mol of O2/mol of substrate, produced 1mol of acid and led to the formation of formate and a new compound with an extinction maximum of 285nm (Compound I). This step was believed to be mediated by a new labile dioxygenase (t½=4h at pH7.0, 4°C) cleaving the pyridine ring between C-2 and C-3. 4. Many of the properties of this pyridine-3,4-diol dioxygenase paralleled those of the extradiol (‘meta’) oxygenases of aromatic-ring cleavage. The extreme lability of the enzyme has so far precluded extensive purification. 5. Compound I showed changes in the u.v.-absorption spectrum with pH but after acidification it was converted into a new product, 3-formylpyruvate, with an extinction maximum now at 279nm. 6. Both Compound I and 3-formylpyruvate were metabolized by extracts but at very different rates. The slower rate of metabolism of Compound I was nevertheless consistent with that of pyridine-3,4-diol metabolism. 7. On acidification Compound I released about 0.65mol of NH3 and has been identified as 3-formiminopyruvate. 8. 3-Formylpyruvate was hydrolysed to formate and pyruvate (Km 2μm) by an acylpyruvate hydrolase active against several other dioxo homologues. The activity of this enzyme was much lower in extracts of succinate-grown cells.
The effect of starvation on insulin secretion and glucose metabolism in mouse pancreatic islets
Authors: Hedeskov Carl J. , Capito Kirsten
Published: August 2015

1. Rates of insulin secretion, glucose utilization, lactate output, incorporation of glucose into glycogen, contents of glucose 6-phosphate, fructose 1,6-diphosphate and ATP, and maximally extractable enzyme activities of hexokinase, high-Km glucose-phosphorylating activity (`glucokinase'), glucose 6-phosphatase and unspecific acid phosphatase were measured in isolated pancreatic islets from fed and 48-h-starved mice. 2. In the fed state insulin secretion from isolated islets was increased five- to six-fold when the extracellular glucose concentration was raised from 2.5mm to 16.7mm; 5mm-caffeine potentiated this effect. The secretory response to glucose of islets from mice starved for 48h was diminished at all glucose concentrations from 2.5mm up to approx. 40mm. Very high glucose concentrations (60mm and above) restored the secretory response to that found in the fed state, suggesting that the Km value for the overall secretory process had been increased (approx. fourfold) by starvation. Addition of 5mm-caffeine to islets from starved mice also restored the insulin secretory response to 2.5–16.7mm-glucose to normal values. 3. Extractable hexokinase, `glucokinase', glucose 6-phosphatase and unspecific phosphatase activities were not changed by starvation. 4. Glucose utilization and glycolysis (measured as the rate of formation of 3H2O from [5-3H]glucose over a 2h period) was decreased in islets from starved mice at all glucose concentrations up to approx. 55mm. At still higher glucose concentrations up to approx. 100mm, there was no difference between the fed and starved state, suggesting that the Km value for the rate-limiting glucose phosphorylation had been increased (approx. twofold) by starvation. Preparation of islets omitting substrates (glucose, pyruvate, fumarate and glutamate) from the medium during collagenase treatment lowered the glucose utilization measured subsequently at 16.7mm-glucose by 38 and 30% in islets from fed and starved mice respectively. Also the 2h lactate output by the islets at 16.7mm extracellular glucose was diminished by starvation. Incorporation of glucose into glycogen was extremely low, but the rate of incorporation was more than doubled by starvation. 5. After incubation for 30min at 16.7mm-glucose the content of glucose 6-phosphate was unchanged by starvation, that of ATP was increased and the concentration of (fructose 1,6-diphosphate plus triose phosphates) was decreased. 6. Possible mechanisms behind the correlated impairment in insulin secretion and islet glucose metabolism during starvation are discussed.
Amino acid sequence of the N-terminal 139 residues of light chain derived from a homogeneous rabbit antibody
Authors: Jaton Jean-Claude
Published: August 2015

The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody (designated BS-1) to type III pneumococci was determined. A combination of methods involving tryptic cleavage restricted to the 2 arginine residues of the molecule and mild acid hydrolysis of a labile peptide bond between the V (variable) and C (constant) regions of the L chain (Fraser et al., 1972) allowed the isolation of two large peptides comprising the entire V region (residues 1–109); these peptides were suitable for automated Edman degradation. The complete sequence analysis of the V region was carried out with only 4μmol of L chain. This material was homogeneous, although minor variant sequences, if present at the 10% value, would not have been detected. The L chain contains 3 intrachain disulphide bridges, whose pairing was established by diagonal electrophoresis: there is one V-region bridge between positions 23 and 88 and one C-region bridge between positions 134 and 194; the third one connects V and C domains between positions 80 and 171. When compared with the basic sequence of human κ chains, rabbit L chain BS-1 appears to be more similar to the VKI prototype sequence than to VKII or VKIII sequences, where VKI, VKII and VKIII represent subgroups I, II and III respectively of V regions of κ light chains. The V regions of rabbit heavy and light chains are homologous to each other. The presence of two clusters of 3 glycine residues in positions 94–96 and 99–101 respectively is remarkable. Residues 94–96 may be related to antibody complementarity whereas residues 99–101 function probably as a pivot permitting the combining region of the L chain to make optimal contact with the antigenic determinant (Wu & Kabat, 1970).
The alkali-labile linkage between keratan sulphate and protein
Authors: Hopwood John J. , Robinson H. Clem
Published: August 2015

Keratan sulphate was isolated from adult intervertebral disc in 90% yield by sequential digestion of the whole tissue with papain, Pronase and Proteus vulgaris chondroitin sulphate lyase. Treatment of this preparation with alkali cleaved a glycosidic bond between N-acetylgalactosamine and threonine and produced, by an alkali-catalysed ‘peeling’ reaction, an unsaturated derivative of N-acetylgalactosamine which reacted as a chromogen in the Morgan–Elson reaction, but remained covalently bonded to the keratan sulphate chain. This derivative was reduced and labelled by alkaline NaB3H4. The substituent at position 3 of N-acetylgalactosamine in the keratan sulphate–protein linkage was identified as a disaccharide, N-acetylneuraminylgalactose, which was isolated from the reaction mixture after alkali treatment.
Kinetics of carbon monoxide binding and electron transfer by cytochrome c polymers
Authors: Dupré Silvestro , Brunori Maurizio , Wilson Michael T. , Greenwood Colin
Published: August 2015

Studies on horse heart cytochrome c polymers were carried out by stopped-flow and photolysis techniques, to investigate the properties of the CO complex and the kinetics of electron transfer, mainly of the dimeric and tetrameric forms. CO binding, which does not occur with native monomers, proceeds at both pH7.0 and pH9.6, and appears to follow complex kinetics: an initial phase is observed, which is CO-concentration-dependent, followed by a very slow monomolecular phase (k~2×10-3s-1 at pH7) before establishment of equilibrium. Photodissociation of the CO complex has a very low quantum yield, probably less than 0.1. Static titration data of the dimer gave an ‘n’ value of 0.4. These data strongly suggest heterogeneity of the population of binding sites, and have been interpreted in terms of the existence of different structures, probably owing to the non-unique type of binding of monomers during polymerization. Polymers of cytochrome c carboxymethylated on the methionine residue normally ligated to iron show simple CO recombination kinetics after photolytic removal (kon=1.5×106m-1·s-1 at pH6). We therefore suggest that, for native cytochrome c, polymerization has an effect on the lability of the haem crevice, rendering the iron available for binding ligands, without, however, forming the structure of a truly open crevice. Electron transfer is, on the other hand, a simple process, and no gross differences are observed between monomer and polymers. A simple model, taking into account all these data, is suggested.
The primary structure of the toxin Laticauda semifasciata III, a weak and reversibly acting neurotoxin from the venom of a sea snake, Laticauda semifasciata
Authors: Maeda Nobuyo , Tamiya Nobuo
Published: August 2015

A weak and reversibly acting neurotoxic protein of Laticauda semifasciata venom, Laticauda semifasciata III (component LsIII), was sequenced. Component LsIII consists of 66 amino acid residues and has five disulphide bridges, one of which was located between residues 26 and 30. The weak and reversible neurotoxicity of component LsIII is discussed in relation to its structure, which falls between those of the neuro- and cardiotoxins of sea snakes and Elapidae snakes isolated and sequenced so far.
Interactions of an intact proteoglycan and its fragments with basic homopolypeptides in dilute aqueous solution
Authors: Gelman Robert A. , Blackwell John , Mathews Martin B.
Published: August 2015

The interactions between a proteoglycan and cationic polypeptides have been investigated by the use of circular-dichroism spectroscopy. The interaction produces an induced conformational change for poly(l-arginine) and poly(l-lysine), similar to the effects previously reported for mucopolysaccharide–polypeptide mixtures. For bovine nasal septum proteoglycan, the interactions are similar to those for chondroitin 4-sulphate, which comprises approximately 63% of the total polysaccharide. The results also suggest that the interactions produce a conformational change in the protein core. Similar studies for the Smith-degradation product show that the protein core can adopt a substantial α-helical content and is capable of interactions with poly-(l-arginine). The interactions for chondroitin sulphate ‘doublets’ are significantly different from those for the separated chains, indicating that the arrangement of the polysaccharide side chains in pairs (and larger groups) along the protein backbone contributes to the interaction properties of the intact proteoglycan.
Properties of the phosphonomethyl isosteres of two phosphate ester glycolytic intermediates
Authors: Stribling Donald
Published: August 2015

The enzymic synthesis of the 1-phosphonomethyl isostere of fructose 1,6-diphosphate in which the 1-phosphate (–OPO3H2) is replaced by the phosphonomethyl group (–CH2PO3H2) is described. The kinetic properties of this fructose diphosphate isostere and of 4-hydroxy-3-oxobutylphosphonic acid, an isostere of dihydroxyacetone phosphate, with aldolase (EC 4.1.2.13), fructose diphosphatase (EC 3.1.3.11) and glycerol phosphate dehydrogenase (EC 1.1.1.8) are described (see Table 1).
Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids
Authors: Whitehouse Sue , Cooper Ronald H. , Randle Philip J.
Published: August 2015

1. Monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, 2-chloropropionate, 2,2′-dichloropropionate and 3-chloropropionate were inhibitors of pig heart pyruvate dehydrogenase kinase. Dichloroacetate was also shown to inhibit rat heart pyruvate dehydrogenase kinase. The inhibition was mainly non-competitive with respect to ATP. The concentration required for 50% inhibition was approx. 100μm for the three chloroacetates, difluoroacetate and 2-chloropropionate and 2,2′-dichloropropionate. Dichloroacetamide was not inhibitory. 2. Dichloroacetate had no significant effect on the activity of pyruvate dehydrogenase phosphate phosphatase when this was maximally activated by Ca2+and Mg2+. 3. Dichloroacetate did not increase the catalytic activity of purified pig heart pyruvate dehydrogenase. 4. Dichloroacetate, difluoroacetate, 2-chloropropionate and 2,2′-dichloropropionate increased the proportion of the active (dephosphorylated) form of pyruvate dehydrogenase in rat heart mitochondria with 2-oxoglutarate and malate as respiratory substrates. Similar effects of dichloroacetate were shown with kidney and fat-cell mitochondria. Glyoxylate, monochloroacetate and dichloroacetamide were inactive. 5. Dichloroacetate increased the proportion of active pyruvate dehydrogenase in the perfused rat heart, isolated rat diaphragm and rat epididymal fat-pads. Difluoroacetate and dichloroacetamide were also active in the perfused heart, but glyoxylate, monochloroacetate and trichloroacetate were inactive. 6. Injection of dichloroacetate into rats starved overnight led within 60 min to activation of pyruvate dehydrogenase in extracts from heart, psoas muscle, adipose tissue, kidney and liver. The blood concentration of lactate fell within 15 min to reach a minimum after 60 min. The blood concentration of glucose fell after 90 min and reached a minimum after 120 min. There was no significant change in plasma glycerol concentration. 7. In epididymal fatpads dichloroacetate inhibited incorporation of14C from [U-14C]glucose, [U-14C]fructose and from [U-14C]lactate into CO2 and glyceride fatty acid. 8. It is concluded that the inhibition of pyruvate dehydrogenase kinase by dichloroacetate may account for the activation of pyruvate dehydrogenase and pyruvate oxidation which it induces in isolated rat heart and diaphragm muscles, subject to certain assumptions as to the distribution of dichloroacetate across the plasma membrane and the mitochondrial membrane. 9. It is suggested that activation of pyruvate dehydrogenase by dichloroacetate could contribute to its hypoglycaemic effect by interruption of the Cori and alanine cycles. 10. It is suggested that the inhibitory effect of dichloroacetate on fatty acid synthesis in adipose tissue may involve an additional effect or effects of the compound.
A steady-state kinetic model of butyrylcholinesterase from horse plasma
Authors: Augustinsson Klas-Bertil , Bartfai Tamas , Mannervik Bengt
Published: August 2015

The steady-state kinetics of the butyrylcholinesterase-catalysed hydrolysis of butyrylthiocholine and thiophenyl acetate were shown to deviate from Michaelis–Menten kinetics. The ‘best’ empirical rate law was selected by fitting different rate equations to the experimental data by non-linear regression methods. The results were analysed in view of two alternative interpretations: (1) the reaction is catalysed by a mixture of enzymes, or (2) the activity is due to a single enzyme displaying deviations from Michaelis–Menten kinetics. It was concluded that the second alternative applies, and this conclusion was further supported by experiments involving simultaneous hydrolysis of alternative thiol ester substrates (butyrylthiocholine/thiophenyl acetate) as well as alternative thiol ester and oxygen ester substrates (butyrylthiocholine/phenyl acetate; thiophenyl acetate/butyrylcholine; acetylthiocholine/phenyl acetate). On the basis of the conclusion that a single enzyme is responsible for the activity, a molecular model is proposed. This model involves an acylated enzyme, and implies binding to the enzyme of one acyl group and one ester molecule, but not two ester molecules at the same time. Thus butyrylcholinesterase, which is structurally a tetramer, behaves functionally as a co-operative dimer, an interpretation in accordance with available data from active-site titrations.
A set of surface proteins common to the circulating human platelet and lymphocyte
Authors: Tanner M. J. A. , Boxer D. H. , Cumming Jane , Verrier-Jones J.
Published: August 2015

The surface proteins of the circulating human platelet and lymphocyte were labelled by using the lactoperoxidase iodination method. Polyacrylamide-gel electrophoresis showed that four corresponding labelled proteins are found on the surface of each cell type. The most intensely labelled protein contains little or no carbohydrate, but the remaining labelled proteins are all glycoproteins. The major labelled band from each cell was isolated and comparative peptide ‘maps’ showed that the two proteins are closely similar. The surface proteins of the lymphocyte and platelet are distinct from those on the erythrocyte, the remaining major type of circulating cell.
Properties of pyruvate dehydrogenase of rat mammary tissue and its changes during pregnancy, lactation and weaning
Authors: Coore Haldane G. , Field Barbara
Published: August 2015

Pyruvate dehydrogenase of rat mammary tissue showed many of the regulatory properties of the analogous enzyme in other mammalian tissues. It was inactivated in the presence of low concentrations of ATP and this rate of inactivation was slowed if pyruvate or PP1 was also present. Reactivation by Mg2+ in the presence of low concentrations of Ca2+ occurred over a similar time-course. The Km value for Mg2+ in this process was about 2mm. The enzyme was assayed in extracts of freeze-clamped mammary glands removed from pregnant, lactating or recently weaned rats under halothane anaesthesia. Both the initial activity and the activity after full activation (‘total enzyme activity’) were determined. The former parameter, when expressed on a DNA basis, varied within a range of 40 times its lowest value. Maximum total enzyme activity was about 1 unit/g wet wt. The total enzyme activity and the fraction in the active form increased in step from pregnancy to mid-lactation, remained elevated until the end of lactation and then fell steeply within 3 days after weaning. The correlation of these two parameters of enzyme activity may indicate a common regulatory factor or else an interdependence arising from inherent properties of the multi-enzyme complex.
Increase in pituitary adenosine 3′:5′-cyclic monophosphate content and potentiation of growth-hormone release from heifer anterior pituitary slices incubated in the presence of 3-isobutyl-1-methylxanthine
Authors: Schofield J. George , McPherson Margaret
Published: August 2015

The release of growth hormone from heifer anterior pituitary slices and the cyclic AMP content of the slices were increased by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, both increases being related to inhibitor concentration over the range 0.1–1.0mm. Neither Ba2+(6.9 or 2.3mm), K+(72mm), nor p-chloromercuribenzoate (20μm) had any effect on pituitary cyclic AMP content over a 20min period. 3-Isobutyl-1-methylxanthine potentiated the release of growth hormone in response to Ba2+(2.3mm) and K+(24mm), but the degree of potentiation did not depend on inhibitor concentration in the same way as did tissue cyclic AMP content. 3-Isobutyl-1-methylxanthine decreased the concentration of K+required to give maximum stimulation of growth-hormone release, but did not significantly increase the maximum response to Ba2+. Growth-hormone release in the presence of prostaglandin E2 (1μm) was increased by 3-isobutyl-1-methylxanthine and was inhibited by the prostaglandin antagonist, 7-oxa-13-prostynoic acid, although this antagonist increased the pituitary cyclic AMP concentration and potentiated the prostaglandin E2-induced rise in cyclic AMP content. The stimulation of growth-hormone release by p-chloromercuribenzoate was not potentiated by 3-isobutyl-1-methylxanthine. The data suggest that Ba+and K+act at the same point in the secretory process as 3-isobutyl-1-methylxanthine, although by a different mechanism, and that p-chloromercuribenzoate has a different point of action.
Regulation of guanylate cyclase in guinea-pig islets of Langerhans
Authors: Howell Simon L. , Montague William
Published: August 2015

1. Guanylate cyclase activity was determined in homogenates of guinea-pig islets of Langerhans by measurement of the conversion of [α-32P]GTP into cyclic [32P]GMP, the reaction products being separated on columns of neutral alumina. 2. The pH optimum of the enzyme was 7.3; it showed a requirement for bivalent cations, the effectiveness of the cations tested being Mn2+»Ca2+>Mg2+. 3. About 70% of enzyme activity was sedimented by centrifugation at 105000g for 60min; activity was increased 2.3-fold by treatment of homogenates with 0.1% Triton X-100. 4. Guanylate cyclase activity of homogenates was increased by acetylcholine, secretin or pancreozymin, but was inhibited by adrenaline, noradrenaline or ATP. Insulin, glucagon, prostaglandins E1 or E2, glucose, F-, diazoxide or glibenclamide were ineffective. 5. Determination of cyclic GMP amounts in islets by radioimmunoassay showed a basal concentration of 2.0pmol/mg of protein, which was increased by incubation of the islets in the presence of acetylcholine or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, but was unaffected by glucose. 6. Dibutyryl cyclic GMP had significant stimulatory effects on rates of insulin biosynthesis in isolated rat islets of Langerhans. 7. These results suggest a possible role for cyclic GMP in the regulation of insulin biosynthesis and secretion.
Sterol biosynthesis by the sea urchin Echinus esculentus
Authors: Smith Andrew G. , Goad L. John
Published: August 2015

1. The 4-demethyl sterols of Echinus esculentus consisted of cholesterol as the major component, with lower concentrations of nine other C26, C27, C28 and C29 Δ5sterols. 2. [2-14C]Mevalonic acid was readily incorporated by the urchin into squalene, lanosterol and desmosterol but only to a small extent into cholesterol. 3. [26-14C]Desmosterol did not appear to be reduced to give cholesterol, but conversion of 5α-[2-3H2]lanost-8-en-3β-ol into cholesterol was observed. 4. No C-24 dealkylation of [4-14C]sitosterol or metabolism of [4-14C]cholesterol could be detected.
Calcium ion transport by pig erythrocyte membrane vesicles
Authors: Buckley J. Thomas
Published: August 2015

Preincubating pig erythrocyte membranes with ATP enhances their ability to accumulate Ca2+against a concentration gradient. The extent of this increase is dependent on preincubation time over the period 0–60min. As the accessibility of outside membrane markers is decreased by preincubation and as accumulated Ca2+is not removed by EGTA [ethanedioxybis(ethylamine)tetra-acetate], it is suggested that ATP causes the formation of sealed inside-out vesicles which can transport Ca2+inward. The transport system requires ATP and Mg2+and exhibits an apparent dissociation constant for Ca2+of approx. 100μm. Since the dissociation constant for Ca2+-sensitive ATPase (adenosine triphosphatase) in these preparations is similar, it is concluded that this ATPase is responsible for Ca2+transport. Polyphosphoinositide concentrations are also increased during incubation with ATP; however, there is no change in their rate of synthesis or breakdown during Ca2+transport.
The estimation of rates of utilization of glucose and ketone bodies in the brain of the suckling rat using compartmental analysis of isotopic data
Authors: Cremer Jill E. , Heath Dennis F.
Published: August 2015

The brains of 18-day-old rats utilize glucose and ketone bodies. The rates of acetyl-CoA formation from these substrates and of glycolysis were determined in vivo from the labelling of intermediary metabolites after intraperitoneal injection of d-[2-14C]glucose, l(+)-[3-14C]- and l(+)-[U-14C]-lactate and d(−)-3-hydroxy[14C]butyrate. Compartmental analysis was used in calculating rates to allow for the rapid exchange of blood and brain lactate, the presence in brain of at least two pools each of glucose and lactate, and the incomplete equilibration of oxaloacetate with aspartate and of 2-oxoglutarate with glutamate. Results were as follows. 1. Glucose and ketone bodies labelled identical pools of tricarboxylate-cycle metabolites, and were in every way alternative substrates. 2. The combined rate of oxidation of acetyl-CoA derived from pyruvate (and hence glucose) and ketone bodies was 1.05μmol/min per g. 3. Ketone bodies contributed 0.11–0.53μmol/min per g in proportion to their concentration in blood, with a mean rate of 0.30μmol/min per g at 1.24mm. 4. Pyruvate and ketone bodies were converted into lipid at 0.018 and 0.008μmol/min per g respectively. 5. Glycolysis, at 0.48μmol/min per g, was more rapid in most rats than pyruvate utilization by oxidation and lipid synthesis, resulting in a net output of lactate from brain to blood. 6. Rates of formation of brain glutamate, glutamine and aspartate were also measured. Further information on the derivation of the models has been deposited as Supplementary Publication SUP 50034 (18 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.
Stimulation by 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and glucocorticoids of phosphoenolpyruvate carboxykinase in the isolated perfused rat liver (Short Communication)
Authors: Huttner Wieland B. , Krone Wilhelm , Seitz Hans J. , Tarnowski Wolfgang
Published: August 2015

Dibutyryl cyclic AMP stimulated the activity of phosphoenolpyruvate carboxykinase in perfused livers of rats, fed on a low-protein diet, linearly over a 6h period. The enzyme activity was also significantly elevated by dexamethasone, the effect being considerably lower than that of the cyclic nucleotide. Since the time-course of phosphoenolpyruvate carboxykinase activity in response to dibutyryl cyclic AMP resembled that observed after dibutyryl cyclic AMP injection into intact animals, it is suggested that induction of the enzyme in vivo is due to a direct action of the cyclic nucleotide on the liver. Combined administration of dibutyryl cyclic AMP and glucocorticoids did not lead to an additive increase of liver phosphoenolpyruvate carboxykinase activity, either in vivo or in the perfused organ.
A study of the coenzyme-binding characteristics of rabbit muscle l-glycerol 3-phosphate dehydrogenase
Authors: Bentley Philip , Dickinson F. Mark
Published: August 2015

1. The binding of NAD+and NADH to glycerol 3-phosphate dehydrogenase was studied in the pH range 6.0–9.0 at 25°C and in the temperature range 16–43°C at pH7.0. 2. The second-order velocity constants for the combination of NADH with the enzyme in the pH range 6.0–9.0 and for the combination of NAD+with the enzyme at pH6.0 were determined. 3. The velocity constant for the dissociation of the enzyme–NAD+complex at pH6.0 was measured.
Isolation of oligomers of 5,6-dihydroxyindole-2-carboxylic acid from the eye of the catfish
Authors: Ito Shosuke , Nicol J. A. Colin
Published: August 2015

The reflecting material of the tapetum lucidum of the sea catfish (Arius felis) was chromatographed on Sephadex LH-20 in methanol–dimethyl sulphoxide–formic acid. Two components were present: one, showing an absorption maximum at 330nm, was tapetal pigment; the other, at 257nm, was an associated nucleoside. The tapetal pigment was extracted in methanol–HCl and isolated by adsorption chromatography on Sephadex LH-20. It yielded a methoxy methyl ester on treatment with diazomethane, and permanganate oxidation gave pyrrole-2,3,5-tricarboxylic acid. From the information provided by u.v. and i.r. spectra of the pigment and its methoxy methyl ester, from elemental analyses and from the oxidation products, we suggest that the tapetal pigment is derived from oxidative coupling of 5,6-dihydroxyindole-2-carboxylic acid. A molecular-weight determination and chromatography of the methoxy methyl ester indicate that the pigment is a mixture of oligomers, among which the tetramers probably predominate. We consider that the monomers are joined mainly by C-C linkages at positions 4 and 7. A synthetic pigment having spectral properties nearly identical with those of the natural pigment was prepared by enzymic oxidation of 5,6-dihydroxyindole-2-carboxylic acid with mushroom tyrosinase. The identity of the tapetal pigment with the synthetic pigment was further confirmed by comparing u.v. and i.r. spectra of their methoxy methyl esters. Formation of the tapetal pigment from tyrosine and relationships of the tapetal pigment to melanin are discussed.
Extraction, partial purification and properties of obelin, the calcium-activated luminescent protein from the hydroid Obelia geniculata
Authors: Campbell Anthony K.
Published: August 2015

1. The Ca2+-activated luminescent protein obelin was extracted from the hydroid Obelia geniculata. 2. After the addition of a large excess of calcium (greater than 5mm) a peak in the rate of luminescence occurred within 100ms, followed by an exponential decay (k=2.8s−1). The obelin activity (light emitted) was measured by the peak height or by the total number of counts recorded on a scalar in the first 10s after addition of Ca2+. 3. After an overnight extraction in 40mm-EDTA–200mm-Tris–HCl, pH7.0, 7.2×1011 counts were obtained from 186g of wet hydroids. 4. The stability of the crude extracts was dependent on pH, being optimal at pH7.0. 5. Obelin could be purified threefold with a yield of 69% by selecting the protein precipitated between 60%- and 100%-saturated (NH4)2SO4. The precipitate could be stored for at least 6 months as a suspension in 40mm-EDTA+saturated (NH4)2SO4, pH7.0, frozen at −70°C with a recovery of 95–100%. 6. Luminescence was also stimulated by Sr2+. However, obelin appeared to have a lower affinity for Sr2+ than for Ca2+. Mg2+ inhibited Ca2+-activated luminescence. 7. Obelin could be used to assay as little as 50pmol of Ca2+ in a final volume of 1ml. 8. At pH7.0 in Ca2+–EGTA [ethanedioxybis(ethylamine)tetra-acetate] buffers the rate of obelin luminescence was proportional to the square of the free Ca2+ concentration in the presence and absence of 1 and 10mm-Mg2+. Over the range 0.1–10μm-Ca2+ less than 0.03% of the obelin was consumed/s. 9. In order to use obelin to study free ionized Ca2+ concentrations similar to those found inside cells in the presence of 10mm-Mg2+ a minimum of 108 counts were required. A total of 1012 counts can be readily extracted from about 200g of wet hydroids. Thus a sufficient quantity of an aequorin-like calcium-activated luminescent protein should now be available to workers in the United Kingdom in order to carry out physiological experiments.
Temperature and pressure adaptation of the binding site of acetylcholinesterase
Authors: Hochachka Peter W.
Published: August 2015

1. Studies with a carbon substrate analogue, 3,3-dimethylbutyl acetate, indicate that the hydrophobic contribution to binding at the anionic site of acetylcholinesterase is strongly disrupted at low temperatures and high pressures. 2. Animals living in different physical environments circumvent this problem by adjusting the enthalpic and entropic contributions to binding. 3. An extreme example of this adaptational strategy is supplied by brain acetylcholinesterase extracted from an abyssal fish living at 2°C and up to several hundred atmospheres of pressure. This acetylcholinesterase appears to have a smaller hydrophobic binding region in the anionic site, playing a measurably decreased role in ligand binding.
Expressions of the Michaelis–Menten equation when studying enzyme reactions in a variable-volume medium (Short Communication)
Authors: Gingold Marcel P.
Published: August 2015

Expressions of integrated rate equations for enzyme reactions studied in a system where the volume of the medium changes, e.g. with a pH-stat device, are given.
The steady-state kinetics of isotope exchange at equilibrium: one substrate–one product enzymic mechanisms where two molecules of substrate or product are bound to an enzyme molecule (Short Communication)
Authors: Darvey Ivan G.
Published: August 2015

The conclusion that the steady-state kinetics of isotope exchange at equilibrium do not show first-order behaviour for some one substrate–one product enzymic mechanisms in which two molecules of substrate or product can be combined with an enzyme molecule at the one time was shown to be erroneous.
Rat cerebral-cortex adenosine deaminase activity and its subcellular distribution
Authors: Pull Ian , McIlwain Henry
Published: August 2015

A radioisotopic assay for adenosine deaminase (EC 3.5.4.4) is described together with its application in investigating the activity of the enzyme in rat cerebral cortex. Activity of the adenosine deaminase was determined to be 115nmol/min per g of tissue, measured in isoosmotic sucrose dispersions of the neocortex, and to be 170nmol/min per g of tissue after treatment with Triton X-100. The enzyme was concluded to be largely cytoplasmic, with a Km of 54–57μm for adenosine. Action of the deaminase, and other aspects of the metabolism of adenosine in intact neocortical tissue, were quantitatively appraised on the basis of the newly determined characteristics.
Urate synthesis in the perfused chick liver
Authors: Barratt E , Buttery P J , Boorman K N
Published: August 2015

Urate synthesis was studied in a perfused chicken liver preparation. The perfused liver had an ATP/ADP ratio of 0.29±0.05(6) compared with 0.34±0.07(10) in liver obtained from chicks under ether anaesthesia. Lactate/pyruvate ratios were 9.4±1.7(5) in the perfused liver and 14.8±1.8(5) in the rapidly sampled liver. Urate synthesis was only marginally stimulated by glycine, glutamine, aspartic acid or NH4Cl, but significant increases were observed with phosphoribosyl pyrophosphate, aminoimidazolecarboxylic acid riboside, inosine, inosinic acid and xanthine. Urate synthesis from glycine, glutamine, NH4Cl, asparagine, alanine, histidine and a mixture of 21 amino acids was obtained on inclusion of insulin in the perfusion medium. Evidence for the inclusion of the carbon of histidine into uric acid was obtained. Aspects of the energy consumption associated with the conversion of excess of amino acid into uric acid are considered.
Albumin and transferrin synthesis during development in the rat
Authors: Yeoh G C T , Morgan E H
Published: August 2015

In this study, the incorporation of [14C]leucine into albumin and transferrin in early rat foetuses, vitelline plus amniotic membranes, chorioallantoic placenta and perinatal rat liver slices was measured and used to detect and compare the rates of synthesis of the two proteins. Albumin synthesis was detected in the body of foetuses from 13 days gestation onwards. Transferrin synthesis was detected only after day 15. Transferrin synthesis was demonstrable in the membranes but not in the chorioallantoic placenta of all the animals investigated, i.e. from 13 to 19 days gestation. Synthesis of albumin and transferrin by the liver of near-term and postnatal animals was shown to correlate with published data on the parenchymal cell number/unit wet wt. of liver. Near-term foetuses synthesized relatively more transferrin than albumin when compared with 10-day postnatal animals. The serum concentrations of the two plasma proteins were also determined. These increased before term whereas the rate of synthesis of albumin and transferrin declined. Postnatally, plasma albumin concentration increased but transferrin concentration decreased, yet the rates of synthesis of both proteins by the liver increased with age. This lack of correlation between the rates of synthesis of the two proteins and their respective plasma concentrations could be explained in part by their increased stability after birth. There was also evidence that the liver haemopoietic cells took up transferrin although they do not synthesize the protein. Thus the decrease in this population of cells during development could also contribute to the discrepancy between liver synthesis and serum concentrations of transferrin.
Transport of catecholamines by resealed chromaffin-granule ‘ghosts’
Authors: Phillips J H
Published: August 2015

A method is described for the preparation of resealed chromaffin-granule ‘ghosts’. The lysis and rapid purification procedures provide ‘ghosts’ in approximately 70% yield from crude granules; the preparation contains 0.1μmol of catecholamine/mg of protein (as compared with 2.8μmol/mg in unlysed granules), of which about one third is inside the ‘ghosts’. The ‘ghosts’ retain their ability to accumulate catecholamines, a process dependent on Mg-ATP and inhibited by reserpine, and a simple assay for this transport is described.
Short-term radiorespirometry of cell suspensions
Authors: Duncombe W G
Published: August 2015

1. An apparatus and method are described with which the oxidation of labelled substrates to14CO2 by cell suspensions may be examined. 2. The use of high-specific-radioactivity substrates at low concentration together with frequent quantitative collection of CO2 permit a more detailed analysis of the appearance of various substrate carbon atoms as CO2 than is possible by existing techniques. 3. Typical experiments with various cell types are reported, in which pathways of glucose oxidation are examined.
Virally mediated membrane changes: inverse effects on transport and diffusion (Short Communication)
Authors: Pasternak Charles A. , Micklem Kingsley J.
Published: August 2015

Sendai virus modifies the cell surface in two ways: (a) by inhibiting facilitated transport and (b) by enhancing passive diffusion.
Purification and properties of rat brain pyruvate carboxylase
Authors: Mahan D E , Mushahwar I K , Koeppe R E
Published: August 2015

Rat brain pyruvate carboxylase was purified 2000-fold and some of its properties and kinetic parameters were investigated. The use of (NH4)2SO4 gradient solubilization on a Celite column and precipitation with polyethylene glycol permitted purification to an estimated 20% purity. Except for a few subtle kinetic differences this enzyme is indistinguishable from rat liver pyruvate carboxylase.
Characterization of a plasmin-digest fragment of rabbit immunoglobulin gamma that binds antigen and complement
Authors: Colomb M , Porter R R
Published: August 2015

Rabbit immunoglobulin gamma (IgG) was digested with plasmin after being left for 15 min at pH2.5, 30 degrees C followed by a rapid increase in the pH to 7. The fragment antigen and complement binding (Facb) was isolated and characterized chemically and biologically. Sequence studies showed that the C-terminal quarter of the heavy chain had been removed, the split occurring at a lysine-alanine bond in the sequence Thr-Ile-Ser-Lys-Ala-Arg. The fragment Facb retained the capacity to precipitate with antigen and the precipitate caused activation of the first component of complement of the same order as that of acid-treated IgG. Both Facb and acid-treated IgG showed a fall in complement fixation relative to the native molecule of 30-40%.
The kinetic of degradation of chondroitin of sulphates and hyaluronic acid by chondroitinase form Proteus vulgaris
Authors: Thurston C F , Hardingham T E , Muir H
Published: August 2015

Km and Vmax. were determined for the degradation by chondroitinase of chondroitin 4-sulphate, 4-sulphate-proteoglycna, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid. Degradation of chondroitin 4-sulphate was inhibited by hyaluronic acid but not by keratan sulphate. The results are discussed with regard to the use to the use of chondroitinase as a sleective reagent for the degradation of tissue glycosaminoglycans.
The sialic acid content and isoelectric point of human kininogen
Authors: Londesborough J C , Hamberg U
Published: August 2015

The sialic acid content of highly purified human kininogen was found to be about 8.6 mol/mol(mol.wt. 50,000). The isoelectric point (pH 4.9 +/- 0.2) is much higher than that of bovine low-molecular-weight kininogen, but is close to that expected from the amino acid and sialic acid analyses.
Comparison of β-lactamase II from Bacillus cereus 569/H/9 with a β-lactamase from Bacillus cereus 5/B/6
Authors: Davies R B , Abraham E P , Fleming J , Pollock M R
Published: August 2015

A mutant of Bacillus cereus 5/B, strain 5/B/6, produces a beta-lactamase II-like enzyme but no beta-lactamase I. Beta-lactamases II and II 5/B/6 appear to show a high degree of homology, but there are significant differences in their enzymic properties.
Hydrolysis of diepptide 2-naphthylamides by human muscle enzymes
Authors: Bury A F , Pennington R J
Published: August 2015

Human muscle enzymes that hydrolyse dipeptide 2-naphthylamides were separated by chromatography on DEAE-Sephadex. Evidence was obtained that these substrates are hydrolysed by arylamidase as well as by dipeptidyl aminopeptidases.
Electron transfer between azurin and cytochrone c-551 from Pseudomonas aeruginosa
Authors: Wilson M T , Greenwood C , Brunori M , Antonini E
Published: August 2015

The electron-transfer reaction between azurin and cytochrome c1 isolated from Pseudomonas aeruginosa was investigated by rapid-reaction techniques. Temperture-jump studies clearly reveal two chemical relaxations, the amplitudes of which have ikentical spectral distributions, but relaxation times show different dependencies on reactant concentrations. Stopped experiments also showed complex kinetics. A model is proposed which is consistent with the kinetic and equilibrium data obtained. The central feature of this model is the proposal that two intercenvertible forms of reduced azurin exist in solution, only one of which si able to participate directly in the electron-transfer reaction with cytochrome c-551. Support for the hypothesis that two forms of reduced azurin exist is derived from studies on the electron-transfer reaction between azurin and Pseudomonas cytochrome oxidase. The possible physiological significance of such a situation is discussed.
Fractionation of proteoglycans from bovine corneal stroma
Authors: Axelsson I , Heinegård D
Published: August 2015

Proteoglycans were extracted from bovine corneal stroma with 4M-guanidinum chloride, purified by DEAE-dellulose chromatography (Antonopoulos et al., 1974) and fractionated by precipitation with ethanol into three fractions of approximately equal weight. One of these fractions consisted of a proteoglycan that contained keratan sulphate as the only glycosaminoglycan. In the othertwo fractions proteoglycans that contained chondroitin sulphate, dermatan sulphate and keratan sulphate were present. Proteoglycans which had a more than tenfold excess of galactosaminoglycans over keratan sulphate could be obtianed by further subfractionation. The gel-chromatographic patterns of the glucosaminoglycans before and after digestion with chondroitinase AC differed for the fractions. The individual chondroitin sulphate chains seemed to be larger in cornea than in cartilage. Oligosaccharides, possibly covalently linked to the protein core of the proteoglycans, could be isolated from all fractions. The corneal proteoglycans were shown to have higher protein contents and to be of smaller molecular size than cartilage proteoglycans.
Sensitive analysis of ethanolamine- and serine-containing phosphoglycerides by high-performance liquid chromatography
Authors: Jungalwala F B , Turel R J , Evans J E , McCluer R H
Published: August 2015

A highly sensitive method for the separation and quantitative measurement of phospholipids containing primary amino groups, such as phosphatidylethanolamine, phosphatidylserine and lysophosphatidylethanolamine, is described. The method involves a simple and quantitative derivative formation of the phospholipids containing amino groups to their u.v.-absorbing biphenylcarbonyl derivatives. These have molar extinction coefficients of about 23,000 at 268nm. The phospholipid derivatives are then separated and non-destructively determined by high-performance liquid chromatography. The amino phospholipids containing vinyl ether bonds (plasmalogens) can be determined separately from the diacyl- and alkylacyl-amino phospholipids. The lower limit of detection by high-performance liquid-chromatographic analysis of the phospholipid derivatives is about 10-13pmol or 0.3-0.4ng of phospholipid P. The quantitative range of derivative formation and analysis by high-performance liquid chromatography of the phospholipids containing amino groups was shown to be 10-500nmol. The method was shown to be applicable to the analysis of phospholipids containing amino groups in tissue samples.
Acetyl-Coenzyme A carboxylase. Role of the prosthetic group in enzyme polymerization
Authors: Landman A D , Darkshinamurti K
Published: August 2015

Apo-(acetyl-CoA carboxylase) completely free from the holoenzyme was prepared from biotin-deficient rat adipose tissue by using affinity chromatography. The apoenzyme does not aggregate under conditions favouring the transition of the holoenzyme to the polymeric form. Such transition is possible after the conversion of the apoenzyme into the holoenzyme in vitro, thus demonstrating the requirement of the prosthetic biotinyl group for enzyme activation.
Drug induction of hepatic glutathione S-transferases in male and female rats
Authors: Kaplowitz N , Kuhlekamp J , Clifton G
Published: August 2015

The induction of the glutathione S-transferases by phenobarbital and polycyclic hydrocarbons was studied in male and female rats. Administration of phenobarbital resulted in 60-80% increase in S-aryl and S-aralkyl enzyme specific activities, whereas the S-epoxide and S-alkyl activities were increased by 30-40%. In following the sequence of induction, the former two activities were noted to reach peak activities before an increase in the latter two activities was observed. Both 3-methylcholanthrene and 3,4-benzopyrene were shown toi nduce these four enzymic activities, although without the discrimination between pairs of activities noted with phenobarbital. No change in Km accompanied the increase in Vmax. after induction by drugs, and no change occurred in Ki for sulphobromophthalein inhibition. Significantly lower enzyme specific activities were found for three of the activities studied in female rats but no difference was observed in the S-alkyltransferase activity. However, the proportional increase in the enzymic activities in response to phenobarbital was the same in males and females. These studies demonstrate the drug induction of a group of cytosolic drug-metabolizing enzymes as well as the identification of sex differences in these activities.
Thymidine kinase of mouse spleen cells in vivo and in vitro
Authors: Olsen I , Harris G
Published: August 2015

The thymidine kinase activity of mouse spleen cells was found to parallel their DNA-synthesizing ability, both in vivo and in vitro. In the former case, more than 90% of the activity of these cells was found in the cytoplasmic fraction. This activity was labile, as was its template mRNA, compared with the nuclear component. The nuclear enzyme increased during culture of the lymphocytes in vitro. Mitogenic stimulation with concanavalin A resulted not only in a greater enhancement of the nuclear activity, but also in a marked increase in the amount of cytoplasmic enzyme. This effect appeared to be mediated via stabilization of the mRNA for the cytoplasmic component. These differences have been considered, especially with respect to the cellular changes that occurred during culture and mitogenic stimulation.