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

ABSTRACT: 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.

SUBMITTER: Parry MJ

PROVIDER: S-EPMC1198334 | biostudies-other | 1967 Nov

REPOSITORIES: biostudies-other

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

Project description:In normal rat liver hexokinase (EC 2.7.1.1) activity usually accounts for not more than 30% of the total glucose-ATP phosphotransferase activity, the remainder being due to glucokinase (EC 2.7.1.2). In the present work it was found that in normal rat liver the relative activities of these two enzymes were occasionally very different from those usually found even though the total glucose-ATP phosphotransferase activity was within the normal range. In some cases almost the entire glucose-ATP phosphotransferase was accounted for by the low-K(m) enzyme hexokinase. Some properties of this enzyme system are reported. It is suggested that this shift in favour of the low-K(m) enzyme without change in the total glucose-ATP phosphotransferase activity may represent a regulatory mechanism.

| S-EPMC1265217 | biostudies-other

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.

| S-EPMC1271219 | biostudies-other

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

Project description: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.

| S-EPMC1264993 | biostudies-other

The mechanism of the reaction catalysed by adenosine triphosphate-creatine phosphotransferase.

Project description:1. The forward and reverse reactions catalysed by ATP-creatine phosphotransferase have been studied kinetically at pH8.0 in the presence and absence of products, under conditions in which the free Mg(2+) concentration was maintained constant at 1mm. Thus at fixed pH the reaction may be considered as being bireactant and expressed as:MgATP(2-)+creatine(0)right harpoon over left harpoonMgADP(-)+phosphocreatine(2-)2. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with a random mechanism in which all steps are in rapid equilibrium except that concerned with the interconversion of the central ternary complexes, and in which two dead-end complexes (enzyme-MgADP-creatine and enzyme-MgATP-phosphocreatine) are formed. The results are in accord with previous suggestions that the enzyme possesses distinct sites for the combination of the nucleotide and guanidino substrates. 3. Values have been determined for the Michaelis and dissociation constants involved in the combination of each substrate with various enzyme forms. Although these values cannot be regarded as absolute, they appear to indicate that the presence of one substrate on the enzyme enhances the combination of the second substrate. In addition, it would seem that in the formation of the enzyme-MgADP-creatine complex the concentration of one reactant does not affect the combination of the other. This contrasts with the formation of the enzyme-MgATP-phosphocreatine complex, where each reactant hinders the combination of the other.

| S-EPMC1264541 | biostudies-other

Properties and reaction with iodoacetamide of adenosine 5'-triphosphate-creatine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action.

Project description:1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP-Mg(2+) plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP-Mg(2+) plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP-Mg(2+) plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.

| S-EPMC1178955 | biostudies-other

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.

| S-EPMC1206586 | biostudies-other

Brain adenosine 5'-triphosphate-creatine phosphotransferase.

Project description:1. The purification of creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) from ox brain by a method that is quicker, simpler and gives much higher yields than other published procedures is described. 2. Stoicheiometric inhibition studies with iodoacetate showed that the enzyme, like that from muscle, has two reactive thiol groups that are essential for enzyme activity. 3. The amino acid sequence around the essential thiol groups was determined and found to be virtually identical with that in creatine kinases from rabbit and ox muscle, and very similar to that found in arginine kinase; the evolutionary significance of this is discussed. 4. The identification of DNS-amino acids on thin layers of silica gel was found to have, in many cases, distinct advantages over that on polyamide layers.

| S-EPMC1179640 | biostudies-other

Regulatory properties of adenosine triphosphate-L-methionine S-adenosyltransferase of rat liver.

Project description:1. Double-reciprocal plots of the reaction velocity of yeast, rat liver and Escherichia coli ATP-l-methionine S-adenosyltransferases (EC 2.5.1.6) as a function of the l-methionine concentrations (under saturating ATP conditions) demonstrate downward deflexions from linearity for the yeast and E. coli adenosyltransferases and an upward deflexion for the rat liver enzyme. 2. The activities of partially purified preparations of rat liver ATP-l-methionine S-adenosyltransferase are enhanced by low concentrations of non-substrate analogues of l-methionine [e.g. 1-aminocyclopentanecarboxylic acid (cycloleucine) and l-2-amino-4-hexynoic acid], or by inorganic tripolyphosphate, an ATP analogue. When the concentrations of these analogues were raised further, the activity decreased. Double-reciprocal plots became linear in the presence of these modifier analogues. The inhibitions are common to all the l-methionine adenosyltransferases examined, but the activation(s) were only found with rat and mouse liver enzymes and not with enzymes obtained from several other tissues of these or other species. 3. The rate of formation of S-adenosyl-l-methionine bears a sigmoidal relation to the l-methionine concentrations when ATP is saturating. The activating effects of the l-methionine analogues and of tripolyphosphate are observed at low l-methionine concentrations, and become obliterated as the l-methionine concentration is raised. These findings are analysed in terms of various regulatory enzyme models.

| S-EPMC1165787 | biostudies-other

Adenosine 5'-triphosphate-arginine phosphotransferase from lobster muscle.

Project description:1. The amino acid composition of lobster-muscle arginine kinase is given and discussed briefly. 2. The results were used to calculate a partial specific volume of 0.735. 3. The results are compared with the amino acid composition of creatine kinase.

| S-EPMC1264971 | biostudies-other

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.

| S-EPMC1174012 | biostudies-other

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