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

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.     

Cause of subunit heterogeneity in purified rat renal phosphate-dependent glutaminase.

When electrophoresed on polyacrylamide gels in the presence of sodium lauryl sulfate, highly purified rat renal phosphate-dependent glutaminase exhibits subunits which range in molecular weight from 57,000 to 75,000. Peptide mapping of the separated subunits following limited proteolysis in the presence of sodium lauryl sulfate shows that all of the various subunits are related in structure. The glutaminase, immunoprecipitated from Triton X-100-solubilized mitochondria, is composed primarily of subunits which have molecular weights of 83,000. In addition, the series of smaller subunits is generated during storage of the Triton-solubilized glutaminase at 4 degrees C. These results indicate that the heterogeneity of subunit size found in the purified glutaminase results from a noninactivating partial proteolysis of the native form of the enzyme.     

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

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria

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.     

The effect of metabolic acidosis on the synthesis and turnover of rat renal phosphate-dependent glutaminase.

Regulation of the mitochondrial phosphate-dependent glutaminase activity is an essential component in the control of renal ammoniagenesis. Alterations in acid-base balance significantly affect the amount of the glutaminase that is present in rat kidney, but not in brain or small intestine. The relative rates of glutaminase synthesis were determined by comparing the amount of [35S]methionine incorporated into specific immunoprecipitates with that incorporated into total protein. In a normal animal, the rate of glutaminase synthesis constitutes 0.04% of the total protein synthesis. After 7 days of metabolic acidosis, the renal glutaminase activity is increased to a value that is 5-fold greater than normal. During onset of acidosis, the relative rate of synthesis increases more rapidly than the appearance of increased glutaminase activity. The increased rate of synthesis reaches a plateau within 5 days at a value that is 5.3-fold greater than normal. Recovery from chronic acidosis causes a rapid decrease in the relative rate of glutaminase synthesis, but a gradual decrease in glutaminase activity. The former returns to normal within 2 days, whereas the latter requires 11 days. The apparent half-time for glutaminase degradation was found to be 5.1 days and 4.7 days for normal and acidotic rats respectively. These results indicate that the increase in renal glutaminase activity associated with metabolic acidosis is due primarily to an increase in its rate of synthesis. From the decrease in activity that occurs upon recovery from acidosis, the true half-life for the glutaminase was estimated to be 3 days.     

Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.     

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

Liver glutaminase can be solubilized from frozen-and-thawed mitochondria by treatment with phospholipase A2. Solubilization by this technique markedly changes the kinetic properties of the enzyme. The properties of the membrane-bound form of the enzyme are partially restored by adding phosphatidylcholine or phosphatidylethanolamine to the phospholipase extract. It is concluded that the kinetic properties of liver glutaminase are a function of the interaction of this enzyme with membrane phospholipids.     

Phosphate-dependent glutaminase from rat kidney. Cause of increased activity in response to acidosis and identity with glutaminase from other tissues.

Immune serum was prepared against phosphate-dependent glutaminase purified from rat kidney and was used to investigate the cause of increased renal glutaminase activity in acidotic rats. Crude kidney homogenates from acidotic rats exhibited a fourfold greater specific activity for phosphate-dependent glutaminase. The glutaminase was solubilized initially by lyophilization of borate treated mitochondria with a 40–60% recovery and with maintenance of threefold difference in specific activity. Both preparations showed the same equivalence point in a quantitative precipitin experiment. To confirm these results, phosphate-dependent glutaminase was also solubilized by treatment of mitochondria isolated from normal and acidotic rat kidney cortex with 1% Triton X-100. The two preparations exhibited a fivefold difference in specific activity and again showed the same equivalence point in a quantitative precipitin experiment. These results indicate that the cause of increased phosphate-dependent glutaminase activity during acidosis is due to the presence of an increased amount of this enzyme. The antiserum prepared against the kidney phosphate-dependent glutaminase did not crossreact with glutaminase solubilized from rat liver mitochondria. But, rat brain mitochondria do contain a phosphate-dependent glutaminase that is immunologically identical to the enzyme from rat kidney.     

The effect of acetyl-coenzyme A on phosphate-activated glutaminase from pig kidney and brain

Phosphate-activated glutaminase (EC 3.5.1.2; l-glutamine amidohydrolase) purified from pig kidney and brain is activated by CoA and short-chain acyl-CoA derivatives. Acetyl-CoA is the most powerful activator (K(A) about 0.2mm). Acetyl-CoA is maximally effective in the absence of other activating anions such as phosphate and citrate, and at low glutamine concentrations. The negative co-operative substrate activation observed at pH7 becomes more pronounced in the presence of acetyl-CoA. Similarly to phosphate, acetyl-CoA produces at high protein concentrations a different type of activation, which is time-dependent, depends on protein concentration and is accompanied by an increase in the sedimentation coefficient. Acetyl-CoA, phosphate and citrate appear to have binding sites in common. No significant difference was observed between kidney and brain phosphate-activated glutaminase.     

Subcellular localization of rat kidney phosphate independent glutaminase

The phosphate independent glutaminase is contained in the brush border membrane of the rat kidney proximal tubule cells. This glutaminase activity cofractionates with the brush border membrane marker activities, alkaline phosphatase and γ-glutamyltranspeptidase, during differential centrifugation. About 30% of these activities are recovered with the mitochondrial fraction, the remainder is pelleted in the heavy microsomal fraction. The phosphate independent glutaminase in both fractions bands, during isopycnic centrifugation, with a mean density of 1.16–1.17 and is coincident with both brush border membrane marker activities. The isolation of intact, individual kidney cells was accomplished by initial perfusion of the kidneys in situ with a collagenase-papain solution followed by a brief incubation in the same enzyme solution. Incubation of isolated cells with a higher concentration of papain results in selective release of the phosphate independent glutaminase. The fact that this occurs without appreciable release of a cytoplasmic marker activity, lactate dehydrogenase, suggests that the phosphate independent glutaminase may be localized on the external surface of the kidney cells.     

Comparison of the hydrolysis and the covalent binding of 6-diazo-5-oxo-L-[6-14C]norleucine by rat renal phosphate-dependent glutaminase

6-Diazo-5-oxo-l-norleucine is both an effective affinity label and a substrate for the rat renal phosphate-dependent glutaminase. Both reactions exhibit a similar phosphate-dependent activation profile. Under the conditions tested, hydrolysis of the diazoketone to yield l-glutamate occurs at a rate approximately 1000-fold greater than the rate of enzyme inactivation. In the presence of phosphate, 6-diazo-5-oxo-l-[6-¹⁴C]norleucine interacts convalently with the glutaminase. Glutamate protects against inactivation and proportionately reduces the extent of [6-¹⁴C]diazoketone binding. The stoichiometry of binding was also proportional to the specific activity of the more labile protomeric form of the glutaminase. With the most active preparation, the normalized stoichiometry approached 1 mol/mol of glutaminase subunit. Tryptic peptide mapping indicates that [6-¹⁴C]diazoketone binding is localized to a single tryptic peptide. These results indicate that 6-diazo-5-oxo-l-[6-¹⁴C]norleucine interacts specifically with a catalytically active group that is located at the glutamine binding site of the phosphate-dependent glutaminase.     

The activity of phosphate-dependent glutaminase from the rat small intestine is modulated by ADP and is dependent on integrity of mitochondria

The effect of adenine nucleotides and phosphate on rat small intestine phosphate-dependent glutaminase (PDG) activity was investigated in intact mitochondria. Disruption of the integrity of mitochondria by sonication or freeze-thawing resulted in loss of enzyme activity. ADP was the strongest adenine nucleotide activator of the enzyme giving a V_max that was over 5-fold of that for AMP or ATP. The sigmoid activation curve of PDG by ADP became hyperbolic in presence ATP. ADP also lowered the K_m for glutamine and increased V_max and these effects were further enhanced by the presence of ATP. Activation of PDG by phosphate and ADP was not completely additive suggesting some antagonism between the activators. There was no clear relationship between changing ATP/ADP ratios and PDG activity in presence of a constant concentration of phosphate. However, ratios of approximately 1:4 and 4:1 gave the highest and lowest activities, respectively. The pH dependence of PDG activity was affected by phosphate concentration and results suggest that the divalent ion is the activating species.     

Inactivation of rat renal phosphate-dependent glutaminase with 6-diazo-5-oxo-L-norleucine. Evidence for interaction at the glutamine binding site.

Inactivation of rat renal phosphate-dependent glutaminase by 6-diazo-5-oxo-L-norleucine occurs only under conditions where the enzyme is catalytically active. The glutaminase activity and the rate of inactivation by the diazoketone exhibit very similar phosphate concentration-dependent activation profiles. Because of this phosphate dependency, it was not possible to differentiate an apparent protection by glutamine from the strong inhibition of inactivation caused by glutamate. The ability of glutamate to protect the glutaminase against inactivation is reversed by increasing concentrations of phosphate.The observed characteristics of inactivation by 6-diazo-5-oxo-L-norleucine differ considerably from those reported for the inactivation by L-2-amino-4-oxo-5-chloropentanoic acid. In addition, the presence of o-carbamoyl-L-serine was found to stimulate inactivation by 6-diazo-5-oxo-L-norleucine, but to protect the glutaminase against inactivation by the chloroketone. Preinactivation of the glutaminase by the diazoketone only slightly reduced the stoichiometry of binding of [5-14C]chloroketone. These observations suggest that 6-diazo-5-oxo-L-norleucine and L-2-amino-4-oxo-5-chloropentanoic acid interact with different sites on the glutaminase which are specific for binding glutamine and glutamate, respectively.     

The maximal activity of phosphate-dependent glutaminase and glutamine metabolism in late-pregnant and peak-lactating rats.

The maximal activity of phosphate-dependent glutaminase was increased in the small intestine, decreased in the liver and unchanged in the kidney of late-pregnant rats. This was accompanied by increases in the size of both the small intestine and the liver. The maximal activity of phosphate-dependent glutaminase was increased in both the small intestine and liver but unchanged in the kidney of peak-lactating rats. Enterocytes isolated from late-pregnant or peak-lactating rats exhibited an enhanced rate of utilization of glutamine and production of glutamate, alanine and ammonia. Arteriovenous-difference measurements across the gut showed an increase in the net glutamine removed from the circulation in late-pregnant and peak-lactating rats, which was accompanied by enhanced rates of release of glutamate, alanine and ammonia. Arteriovenous-difference measurements for glutamine showed that both renal uptake and skeletal-muscle release of glutamine were not markedly changed during late pregnancy or peak lactation; but pregnant rats showed a hepatic release of the amino acid. It is concluded that, during late pregnancy and peak lactation, the adaptive changes in glutamine metabolism by the small intestine, kidneys and skeletal muscle of hindlimb are similar; however, the liver appears to release glutamine during late pregnancy, but to utilize glutamine during peak lactation.