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1.
Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.  相似文献   

2.
Astrocytes in primary cultures contain a relatively high activity, of phosphate activated glutaminase, although it is significantly lower than that of synaptosomal enriched preparations. The relatively high glutaminase activity in the astrocytes appears not to be caused by substrate induction, since a 10-fold variation in the glutamine concentration of the culture medium does not affect the activity. Of the reaction products, only glutamate inhibits astrocytic glutaminase whereas that of synaptosomal enriched preparations is inhibited by both glutamate and ammonia. Similar to the synaptosomal enzyme, glutaminase in astrocytes is inhibited about 50% by N-ethylmaleimide, indicating N-ethylmaleimide-sensitive and-insensitive compartments of the enzyme. Calcium activates glutaminase in astrocytes as in synaptosomes, by promoting phosphate activation. Except for the lower activity and the lack of effect of ammonia, the properties of the astroglial glutaminase has been found to be no different from that of the synaptosomal one. The relatively unrestrained astroglial glutaminase may, however, argue against the concept of a glutamine cycle operating in a stoichiometric manner.Abbreviations NEM N-ethylmaleimide - PAG Phosphate-activated glutaminase - PMB p-mercuribenzoate  相似文献   

3.
Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase.  相似文献   

4.
The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticale sp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH4+). A monovalent anion (Cl-) and a multivalent anion (phosphate) were shown to activate the glutaminase. Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli.  相似文献   

5.
Maple syrup urine disease is a metabolic disorder caused by mutations of the branched chain keto acid dehydrogenase complex, leading to accumulation of alpha-keto acids and their amino acid precursors in the brain. We now report that alpha-ketoisovaleric, alpha-keto-beta-methyl-n-valeric and alpha-ketoisocaproic acids accumulated in the disease inhibit glutamate uptake into rat brain synaptic vesicles. The alpha-keto acids did not affect the electrochemical proton gradient across the membrane, suggesting that they interact directly with the vesicular glutamate carrier. Chloride anions have a biphasic effect on glutamate uptake. Low concentrations activate the uptake (0.2 to 8 mM), while higher concentrations are inhibitory. The alpha-keto acids inhibited glutamate uptake by a new mechanism, involving a change in the chloride dependence for the activation of glutamate uptake. The activation of glutamate uptake by low chloride concentrations was shifted toward higher concentrations of the anion in the presence of alpha-keto acids. Inhibition by alpha-keto acids was abolished at high chloride concentrations (20 to 80 mM), indicating that alpha-keto acids specifically change the stimulatory effect of low chloride concentrations. High glutamate concentrations also reduced the inhibition by alpha-keto acids, an effect observed both in the absence and in the presence of low chloride concentrations. The results suggest that in addition to their possible pathophysiological role in maple syrup urine disease, alpha-keto acids are valuable tools to study the mechanism of vesicular transport of glutamate.  相似文献   

6.
Physiological role of glutaminase activity in Saccharomyces cerevisiae   总被引:1,自引:0,他引:1  
The participation of glutaminase activity in glutamine degradation was studied in a wild-type strain (S288C) of Saccharomyces cerevisiae. Evidence is presented that this strain has two glutaminase activities, a readily extractable form (glutaminase B) and a membrane-bound enzyme (glutaminase A). Glutaminase A and B activities could also be distinguished by their thermostability, pyruvate sensitivity and pH optimum. Glutaminase B activity was negatively modulated by some 2-oxo acids, and in vivo pyruvate accumulation inhibited this activity. A mutant strain (CN10) with an altered glutaminase B activity was isolated and partially characterized. Its glutaminase B activity was more sensitive to inhibition by pyruvate and 2-oxoglutarate than the wild type, thus resulting in inactivation of this enzyme in vivo. The physiological role of glutaminase activity is discussed with regard to the phenotype shown by the mutant strain.  相似文献   

7.
8.
Excretion of alpha-keto acids by clinical isolates and laboratory strains of Salmonella typhimurium was determined by high-performance liquid chromatography analysis of culture supernatants. The levels of excretion increased markedly with increasing iron stress imposed by the presence of alpha,alpha'-dipyridyl or conalbumin in the medium. The major product was pyruvic acid, but significant concentrations of alpha-ketoglutaric acid, alpha-ketoisovaleric acid, and alpha-ketoisocaproic acid were also observed. Maximal excretion occurred at iron stress levels that initially inhibited bacterial growth; the concentration of alpha,alpha'-dipyridyl at which this was observed differed between strains depending on their ability to secrete and utilize siderophores, suggesting that the intracellular iron status was important in determining alpha-keto acid excretion. However, prolonged incubation of the siderophore-deficient S. typhimurium strain enb-7 under conditions of high iron stress resulted in significant delayed bacterial growth, promoted by tonB-dependent uptake of iron complexed with the high accumulated levels of pyruvic acid and other alpha-keto acids. Strain RB181, a fur derivative of enb-7, excreted massive amounts of alpha-keto acids into the culture medium even in the absence of any iron chelators (the concentration of pyruvic acid, for example, was >25 mM). Moreover, RB181 was able to grow and excrete alpha-keto acids in the presence of alpha,alpha'-dipyridyl at concentrations threefold greater than that which inhibited the growth of enb-7.  相似文献   

9.
We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply. The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone. The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers.  相似文献   

10.
Abstract: Phosphate-activated glutaminase (EC 3.5.1.2) in synaptosomal preparations is inhibited 40–60% by the sulphydryl group reagent N -ethylmaleimide (NEM), forming the basis for distinction between NEM-sensitive and NEM-insensitive glutaminases. The NEM effect cannot be explained by differential effects on distinct glutaminases because other glutaminases have not been detected, and the synaptosomal glutaminase activity can be fully accounted for by the activity of phosphate-activated glutaminase. By fractionation of mitochondria isolated from synaptosomal preparations, which are preincubated with and without NEM, both NEM-sensitive and NEM-insensitive glutaminases are found to be localized to the inner mitochondrial membrane. Variations in pH (7.0–7.6) and the phosphate concentration (5–10 mM) affect chiefly NEM-sensitive glutaminase, demonstrating that this glutaminase may be subject to regulation by compounds in the cytosol having restricted permeability to the inner mitochondrial membrane. Since p -hydroxymercuribenzoate, which is known to be impermeable to the inner mitochondrial membrane, inhibits glutaminase similarly to NEM, phosphate-activated glutaminase is assumed to be compartmentalized within the inner mitochondrial membrane. Thus, NEM-sensitive glutaminase is localized to the outer face and NEM-insensitive glutaminase to the inner region of this membrane and probably also to the matrix region.  相似文献   

11.
Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant glutaminases, designated glutaminase I and II. Glutaminase I was homogeneously purified about approximately, 1620-fold with a 4% yield, and was a dimer with a molecular weight of about 86,000. Glutaminase II was partially purified about 190-fold with a 0.04% yield. The molecular weight of glutaminase II was also 86,000. Maximum activity of glutaminase I was observed at pH 8.0, 50°C and 8–16% NaCl. The optimal pH and temperature of glutaminase II were 8.5 and 50°C. The activity of glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of glutaminase I. Both enzymes catalyzed the hydrolysis of l-glutamine, but not its hydroxylaminolysis. The Km values for l-glutamine were 4.4 (glutaminase I) and 6.5 mM (glutaminase II). Neither of the glutaminases were activated by the addition of 2 mM phosphate or 2 mM sulfate. p-Chloromercuribenzoate (0.01 mM) significantly inhibited glutaminase I, but not glutaminase II. The conserved sequences LA**V and V**GGT*A were observed in the N-terminal amino acid sequences of glutaminase I, similar to that for other glutaminases.  相似文献   

12.
Glutamine synthetase and glutaminase activities in various hepatoma cells   总被引:4,自引:0,他引:4  
Glutamine synthetase and glutaminase activities in a series of hepatoma cells of human and rat origins were determined for comparison with normal liver tissues. Marked decrease in glutamine synthetase activity was observed in the tumor cells. Phosphate-dependent and phosphate-independent glutaminase activities were increased compared with those from normal liver tissues. Well coupled mitochondria were isolated from HuH 13 line of human hepatoma cells and human liver. Oxypolarographic tests showed that glutamine oxidation was prominent in the tumor mitochondria, while mitochondria from the liver showed a feeble glutamine oxidation. Glutamine oxidation was inhibited by prior incubation of the mitochondria with DON (6-diazo-5-oxo-L-norleucine), which inhibited mitochondrial glutaminase. These results indicate that the product of glutamine hydrolysis, glutamate, is catabolized in the tumor mitochondria to supply ATP.  相似文献   

13.
The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticalesp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH+ 4). A monocharged anion (Cl) and a multicharged anion (phosphate) were shown to activate the glutaminase. Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli.  相似文献   

14.
Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.  相似文献   

15.
Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.  相似文献   

16.
Temporal inhibition of protein synthesis with cycloheximide prevents subsequent insulin, but not serum-stimulated DNA synthesis in G1-arrested chick embryo fibroblasts (CEF). The inhibition is measured by the incorporation of 3H-thymidine into acid insoluble material and confirmed by chemical estimate of the DNA content of inhibited and uninhibited cells. Cycloheximide treatment is without effect if the cell cultures are maintained at 4 degrees C while exposed to the drug. Several alpha-keto acids (pyruvate, oxaloacetate, alpha-ketobutyrate) at 0.5-1 mM concentrations restore DNA synthesis in previously inhibited cells when combined with insulin. L-alanine (D-alanine is inert) is even more effective than the keto acids in stimulating DNA synthesis after cycloheximide treatment. Glucose transport was unaffected by cycloheximide treatment while lactate levels in medium from inhibited, insulin-stimulated CEF were reduced 70% compared to uninhibited counterparts. We speculate that cycloheximide treatment may lead to the decay of a glycolytic enzyme which compromises the ability of inhibited cells to synthesize pyruvate from glucose, and thus induces an exogenous requirement for alpha-keto acid or L-alanine. A serum component(s) with a molecular weight of about 100 permitted insulin-stimulated DNA synthesis in inhibited cells.  相似文献   

17.
1. In respiring rat liver mitochondria EDTA stimulates glutaminase activity measured in the presence of phosphate and HCO3- ions. The stimulation can be reversed by the addition of low concentrations of MgCl2. EGTA does not stimulate glutamine hydrolysis. 2. Glutaminase activity assayed in disrupted mitochondria is not significantly affected by EDTA or MgCl2. 3. The addition of EDTA results in a decrease in the concentration of phosphate required for half-maximal glutaminase activity. 4. Depletion of mitochondrial Mg2+ by the addition of the ionophore A23187 also stimulates glutamine hydrolysis in both the presence and the absence of EDTA. The effect of the ionophore can be abolished by the addition of MgCl2. 5. Hypo-osmotic incubation conditions increase the rate of mitochondrial glutamine hydrolysis. The effect of hypo-osmoticity on glutaminase is much less when EDTA is present. 6. It is suggested that glutaminase is partially and indirectly inhibited by endogenous mitochondrial Mg2+ and that the inner membrane may play a role in the regulation of glutaminase activity.  相似文献   

18.
ACTIVATION OF PIG BRAIN GLUTAMINASE   总被引:3,自引:2,他引:1  
Pig brain glutaminase (EC 3.5.1.2, l -glutamine amidohydrolase) is activated by certain anions (e.g. phosphate, fluoride, carboxylic acids) and inhibited by others (e.g. chloride, bromide, iodide and glutamate). The only cation which has been found to activate the enzyme is the ammonium ion. This applies to both the tris-HCl form and the phosphate-borate form of glutaminase.  相似文献   

19.
The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.  相似文献   

20.
Two transaminase inhibitors, aminooxyacetate and cycloserine, inhibited the initiation of insulin-stimulated DNA synthesis in chick embryo fibroblasts. This inhibition was overcome when pyruvate (4 mM), oxaloacetate (4 mM), or alpha-ketobutyrate (10 mM) was included in the culture medium with hormone and inhibitor. Aminooxyacetate also inhibited lactate production in insulin-treated cultures in the absence of added alpha-keto acid.  相似文献   

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