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Localization and some properties of phosphate-dependent glutaminase in disrupted liver mitochondria.

ABSTRACT: 1. Glutaminase activity in frozen and thawed liver mitochondria was activated by NH4+, phosphate and HCO3-ions and also by ATP . 2. NH4+ and HCO3-ions decreased the requirement of the enzyme for phosphate. The activation by ATP was observed only in the presence of NH4+ or HCO3-ions. 3. In frozen-and-thawed mitochondria, the enzyme was loosely bound to the inner membrane, the Arrhenius plot showing a break at 23 degrees C. On sonication, glutaminase was detached from the membrane and the Arrhenius plot became linear. 4. The apparent Km for glutamine of the membrane-bound form was 6 mM, and that of the soluble form was 21 mM. 5. It is likely that the properties of glutaminase in the intact cell are dependent on the association of this enzyme with the mitochondrial membrane.

SUBMITTER: McGivan JD

PROVIDER: S-EPMC1162368 | biostudies-other | 1980 Nov

REPOSITORIES: biostudies-other

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Intracellular localization and properties of phosphate-dependent glutaminase in rat mesenteric lymph nodes.

Project description:Phosphate-dependent glutaminase was present at approximately similar activities in lymph nodes from mammals other than rat, and in thymus, spleen, Peyer's patches and bone marrow of the rat. This suggests that glutamine is important in all lymphoid tissues. Phosphate-dependent glutaminase activity was shown to be present primarily in the mitochondria of rat mesenteric lymph nodes, and most of the activity could be released by detergents. The properties of the enzyme in mitochondrial extracts were investigated. The pH optimum was 8.6 and the Km for glutamine was 2.0 mM. The enzyme was activated by phosphate, other phosphorylated compounds including phosphoenolpyruvate, and also leucine: 50% activation occurred at 5, 0.2 and 0.6 mM for phosphate, phosphoenolpyruvate and leucine respectively. The enzyme was inhibited by glutamate, 2-oxoglutarate, citrate and ammonia, and by N-ethylmaleimide and diazo-5-oxo-L-norleucine; 50% inhibition was observed at 0.7 and 0.1 mM for glutamate and 2-oxoglutarate respectively. Some of these properties may be important in the control of the enzyme activity in vivo.

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Influence of phospholipids on the activity of phosphate-dependent glutaminase in extracts of rat liver mitochondria.

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Factors influencing the inactivation of phosphate-dependent glutaminase in the matrix fraction of rat liver mitochondria.

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Increased activity of phosphate-dependent glutaminase in liver mitochondria as a result of glucagon treatment of rats.

Project description:1. Injection of rats with glucagon leads to an increased effective activity of glutaminase in subsequently isolated liver mitochondria. 2. This effect of glucagon is manifested as a decreased requirement of glutaminase for phosphate in the presence of HCO3-. The HCO3--concentration-dependence is unchanged. 3. The effect of glucagon is lost on disruption of the mitochondria. 4. In accordance with previous reports, incubation of mitochondria in hypo-osmotic media also increases the effective activity of glutaminase. Glucagon increases glutamine hydrolysis at intermediate osmolarities of the suspending medium, but does not affect glutaminase activity when it is already maximally activated by hypo-osmotic conditions. 5. From this and previous work, it seems that hypo-osmotic incubation conditions, EDTA and glucagon may all activate glutaminase by a common mechanism. It is postulated that this mechanism involves modification of the interaction of glutaminase with the mitochondrial inner membrane.

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Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

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Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism.

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Glutamine catabolism by heart muscle. Properties of phosphate-activated glutaminase.

Project description:1. Rat heart homogenates catabolized glutamine in the presence of rotenone, an inhibitor of the respiratory chain. 2. The reaction was markedly stimulated by phosphate and inhibited by glutamate, but not greatly affected by ammonia. 3. Glutamine breakdown was enhanced by lactate, malate, citrate and ATP, and almost completely blocked by 1 mM-N-ethylmaleimide. 4. The Km for glutamine measured in homogenates supplemented with either 50 mM- or 100 mM-phosphate and at pH 7.3 or 8.0 was about 4 mM, whereas the Vmax was larger at higher anion concentrations and at the more alkaline pH. 5. Activity of glutaminase was 3-fold greater in isolated mitochondria than in muscle homogenates. Distribution of the activity was the same as that of a mitochondrial marker, cytochrome c oxidase. 6. Properties of glutaminase with respect to its dependence on the concentrations of phosphate, glutamine and H+ were the same in intact and broken mitochondria and very similar to those in homogenates. However, the specific activity of the enzyme was considerably smaller in frozen-thawed than in intact organelles. 7. It is concluded that heart mitochondria possess a kidney-type phosphate-activated glutaminase that can serve as a source of myocardial glutamate.

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Properties of rat renal phosphate-dependent glutaminase coupled to Sepharose. Evidence that dimerization is essential for activation.

Project description:In the absence of phosphate, purified rat renal phosphate-dependent glutaminase exists as a catalytically inactive protomer. The addition of phosphate results in both dimerization and activation of the glutaminase. Covalent attachment of the dimeric form of the glutaminase to CNBr-activated Sepharose was achieved with 84% retention of activity. At least 70% of the bound glutaminase activity was expressed even in the absence of added phosphate. In addition, 6-diazo-5-oxo-L-norleucine, which interacts only with the catalytically active form of the glutaminase, inactivates the bound dimeric form of glutaminase at the same rate in either the absence or the presence of added phosphate. Therefore retention of dimeric structure is apparently sufficient to maintain glutaminase activity. In contrast, the coupling of the protomeric form of the enzyme to Sepharose resulted in retention of only 3% of the phosphate-induced glutaminase activity. However, up to 48% of this activity could be reconstituted by addition of soluble glutaminase under conditions that promote dimerization. These results indicate that the monomeric form of the glutaminase has minimal inherent activity and that dimerization is an essential step in the phosphate-induced activation of the glutaminase.

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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.

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Inhibition by glutamate of phosphate-dependent glutaminase of rat kidney.

Project description:A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.

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