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Purification and properties of adenosine 5'-triphospae-D-glucose 6-phosphotransferase from rat liver.

ABSTRACT: 1. An 870-fold purification of glucokinase from rat liver is described which involves ammonium sulphate fractionation and the use of DEAE-Sephadex, DEAE-cellulose and polyacrylamide columns. 2. The preparation is free of any interfering enzymes and has a specific activity of 8mumoles/min./mg. of protein. 3. Glucokinase catalyses the phosphorylation of glucose, mannose and 2-deoxyglucose. 4. The enzyme is inhibited by high concentrations of glucose 6-phosphate only; ADP is an inhibitor whose effect depends on the Mg(2+) concentration. 5. The properties of glucokinase are compared briefly with those of other phosphotransferases.

SUBMITTER: Parry MJ

PROVIDER: S-EPMC1264993 | biostudies-other | 1966 May

REPOSITORIES: biostudies-other

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Further properties and possibel mechanism of action of adenosine 5'-triphosphate-D-glucose 6-phosphotransferase from rat liver.

Project description:1. Magnesium ions are the most effective bivalent ions in the glucokinase reaction. 2. The molecular weight of rat hepatic glucokinase is 48000-49000 as assessed by gel filtration on Sephadex G-100. 3. Anomalous kinetic behaviour at low glucose concentrations appears to be due to the formation during the purification procedure of fragments possessing modified catalytic properties, but is unlikely to be of physiological significance. 4. Extension of previous studies (Parry & Walker, 1966) suggests that glucokinase catalyses a reaction of the random Bi Bi type similar to that of yeast hexokinase. 5. The inhibitory effects of various thiol reagents suggest that a thiol group may be involved at or near the binding site of the acceptor molecule.

| S-EPMC1198334 | biostudies-other

Some properties of rat-liver glucose--adenosine triphosphate phosphotransferases.

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ADENOSINE 5'-TRIPHOSPHATE-ARGININE PHOSPHOTRANSFERASE FROM LOBSTER MUSCLE: PURIFICATION AND PROPERTIES.

Project description:1. A simple chromatographic method is described for the purification of arginine kinase from lobster (Homarus vulgaris) muscle. 2. Some physical properties and the effects on enzyme activity of ionic strength, pH, buffer salts, metal ions and substrates are reported. 3. The kinetic parameters, evaluated by variation of the concentration of one of the substrates, are dependent on the concentration of the other substrate. 4. The properties of the enzyme are discussed in relation to previous findings about phosphagen phosphotransferases.

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Multiple forms of glucose-adenosine triphosphate phosphotransferase in rat mammary gland.

Project description:Measurements have been made of the total hexokinase activity and of the relative amounts of types I and II hexokinase in rat mammary gland and at different stages of the lactation cycle. The total hexokinase activity increased during lactation, that of type II increasing to a greater extent than that of type I; the type II/type I activity ratio rose from a pregnancy value of about 1 to a mid-lactation value of 3, returning to 1 on involution. The changes in type II hexokinase activity during the lactation cycle parallel the changes in the insulin sensitivity of mammary-gland tissue. A study of the effect of alloxan-diabetes on mammary-gland hexokinase during the mid-lactation period revealed that, although the total glucose-phosphorylating capacity of the mammary gland was almost unchanged, the relative contributions of type I and type II hexokinases altered, decreasing the type II/type I activity ratio to about 1.

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Purification and properties of adenosine triphosphate-creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula.

Project description:1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl(-) restores protection to the original value. The quaternary MgADP-creatine-enzyme-nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on V(max.) and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.

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Rat liver alcohol dehydrogenase. Purification and properties.

Project description:Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.

| S-EPMC1178266 | biostudies-other

Subcellular localization and properties of rat liver adenosine diphosphatase.

Project description:ADPase (adenosine diphosphatase) was assayed in rat liver homogenates with [beta-32P]ADP as substrate. The activity had a pH optimum of 8.0 and was strongly activated by Mg2+. The intracellular localization was determined by analytical subcellular fractionation with single-step sucrose-density-gradient centrifugation. Selective membrane perturbants were used to enhance the resolution of the various organelles. ADPase was localized to the mitochondria. Mitochondria were isolated by differential centrifugation and subfractionated by selective disruption of the inner and outer membranes. The intramitochondrial localization of ADPase was compared with various marker enzymes and was shown to be concentrated in the outer-membrane fractions. The effects of various inhibitors on the ADPase activity were determined and the possibility that the activity could be due to known enzyme systems was considered. It is concluded that ADP degradation is due to a hitherto unrecognized mitochondrial enzyme.

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Partial purification and properties of rat liver glutaminase.

Project description:The mitochondrial enzyme phosphate-dependent glutaminase was partially purified from rat liver. The enzyme had Mr 290 000 as judged by chromatography on Sephacryl S-300. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the preparation, glutaminase was tentatively identified with a peptide of Mr 73 500. The concentration-dependence on glutamine was highly sigmoidal, with half-maximum velocity at 22 mM-glutamine. Half-maximum activity was obtained with 5 mM-phosphate. The enzyme required ammonia as an obligatory activator, in agreement with previous reports on intact and sonicated mitochondria. These findings further differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney and several other tissues.

| S-EPMC1153663 | biostudies-other

Partial purification and properties of rat liver mitochondrial polynucleotide phosphorylase.

Project description:1. Polynucleotide phosphorylase was partially purified from the inner membrane of rat liver mitochondria. 2. The partially purified particulate enzyme catalyses phosphorolysis of poly(A), poly(C), poly(U) and RNA to nucleoside diphosphates. 3. It is devoid of nucleoside diphosphate-polymerization activity. 4. Variable amounts of ADP/P(i)-exchange activity are associated with the polynucleotide phosphorylase and are probably due to a different enzyme. 5. ADP is the preferred substrate for exchange, and little or no reaction occurs with other nucleoside diphosphates, but ATP/P(i)-exchange takes place at one-third the rate observed with ADP. 6. The partially purified enzyme is free from the phosphatases found in the crude mitochondrial inner membrane, but is associated with an endonuclease activity and some adenylate kinase activity; no cytidylate kinase activity analogous to the latter was detectable.

| S-EPMC1174413 | biostudies-other

Purification and some kinetic properties of rat liver glucosamine synthetase.

Project description:1. Glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was purified about 300-fold from rat liver by two techniques. One procedure utilized the protective action of fructose 6-phosphate and gave a relatively stable preparation, the other yielded an unstable enzyme (half-life of about 20h), free of contaminant activities, on which kinetic experiments were performed. Although the properties of the two preparations showed slight differences, the unstabilized form could be converted into the stabilized form. 2. During preparation the enzyme retained its sensitivity to the feedback inhibitor, UDP-N-acetylglucosamine. 3. The reversibility of the enzyme-catalysed reaction could not be demonstrated. There was no apparent requirement for a cofactor. 4. The pH optimum was at 7.5, at which pH the reaction obeyed a Ping Pong Bi Bi rate equation. At pH values outside the range 6.9-7.6 and at temperatures below 29 degrees C the velocity was described by an ordered Bi Bi rate equation. 5. The molecular weight of the enzyme, determined by two procedures, was 360000-400000. 6. The aminotransferase was unable to utilize ammonia as a substrate.

| S-EPMC1176648 | biostudies-other

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