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.     

The purine nucleotide cycle in the regulation of ammoniagenesis during induction and cessation of chronic acidosis in the rat kidney.

The effect of chronic acid feeding and its subsequent withdrawal was determined on the amounts of the metabolic intermediates and enzymic activities of the purine nucleotide cycle. Sprague-Dawley rats were given 1.5% (w/v) NH4Cl in their drinking water for 5 days. The renal excretion of NH3 rose 70-fold and the rats developed acidosis. The amount of renal IMP rose from a control value of 4.5 +/- 2.2 to 20.4 +/- 3.7nmol/g of kidney after 48h of acid feeding (P less than 0.001) and fell to normal within 48h of the recovery. Adenylosuccinate concentrations fell from a control value of 4.5 +/- 0.9nmol/g of kidney to 1.2 +/- 0.3nmol/g (P less than 0.005) by day 5 of acidosis and continued to fall to undetectable values by 48h after recovery. The amount of AMP remained constant through the acid-feeding and the recovery periods. The activity of adenylosuccinate synthetase, the rate-limiting enzyme of the purine nucleotide cycle, paralleled the rise and fall in NH3 excretion. The activities of phosphate-dependent glutaminase and glutamate dehydrogenase were elevated during the acid-feeding and the recovery period. Thus changes in the purine nucleotide cycle correlate with changes in NH3 excretion to a more parallel degree than does the activity of glutaminase or glutamate dehydrogenase.     

Activity of renal enzymes producing ammonia from glutamine in the absence of phosphate: 2. Effect of phosphate dependent glutaminase inhibition by heating

It is known that heating at 50 degrees C for 10 minutes inhibits phosphate dependent glutaminase (PDG) activity of renal cortex, without any effect on gamma-glutamyl transpeptidase (gamma GT) and its phosphate independent glutaminase (PIG) activity. The effect of heating on PIG and total gamma GT activities was evaluated in renal cortex homogenates of rats both in normal acid-base equilibrium and in chronic metabolic acidosis (CMA). Homogenates were incubated in a medium containing glutamine 2 mM, no phosphate, at pH 7,40. PIG activity was measured as glutamate production and total gamma GT activity as ammonia production. In normal rats PIG activity was unchanged after heating, whereas a significant decrease of total gamma GT activity was observed (p less than 0,01). CMA caused an increase in both PIG and total gamma GT activity (p less than 0,01) and these increased to a further extent after heating. In both normal and acidotic rats the glutamate production/ammonia production ratio rose to about 1. In conclusion: a) in the experimental setting used for this study PDG activity does not intervene in glutamate and ammonia production from glutamine; b) heating causes an inhibition of gamma GT activities, other than PIG, both in normal and in acidotic rats; c) in CMA heating increases PIG activity of gamma GT.     

The regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat kidney cortex. The role of acid-base balance and glucocorticoids.

The effects of metabolic acidosis and of hormones on the activity, synthesis, and degradation of renal cytosolic P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) were studied in the rat using isotopic -immunochemical procedures. At normal acid-base balance, the synthesis of the enzyme accounted for between 2 and 3.5% of the synthesis of all soluble protein in the kidney cortex. P-enolpyruvate carboxykinase synthesis was selectively stimulated in acute metabolic acidosis, so that the relative rate of synthesis of the enzyme was increased to 7% 13 hours after oral administration of ammonium chloride. The stimulation of P-enolpyruvate carboxykinase synthesis preceded any increase in the assayable activity of the enzyme. The administration of sodium bicarbonate to acutely acidotic rats returned the rate of enzyme synthesis to normal in 8 hours. The effect of acidosis on both the synthesis and the activity of P-enolpyruvate carboxykinase was prevented by actinomycin D, cordycepin, and cycloheximide. The degradation in vivo of pulse-labeled P-enolpyruvate carboxykinase was not affected by acidosis. Thus, the stimulation of P-enolpyruvate carboxykinase synthesis is the major mechanism for the increase in the level of the enzyme observed in metabolic acidosis. The administration of glucocorticoid triamcinolone resulted in an increase in the relative rate of P-enolpyruvate carboxykinase synthesis and a commensurate increase in the activity of the enzyme in the renal cortex. Both changes were abolished by actinomycin D. Fasting was characterized by a high enzyme activity and a rapid rate of enzyme synthesis in the kidney cortex. This high rate of synthesis was reduced after the administration of sodium bicarbonate, but not after glucose feeding. Moreover, the injection of insulin to diabetic rats did not repress P-enolpyruvate carboxykinase synthesis in the renal cortex. Theophylline plus N-6, 0-2'-dibutyryl adenosine 3':5'-monophosphate stimulated P-enolpyruvate carboxykinase synthesis in the kidney of intact rats. However, the latter effect was probably due to glucocorticoid secretion, since it did not occur in adrenalectomized animals. The administration of parathyroid extracts did not result in the induction of the enzyme. Thus, the hormonal regulation of cytosolic P-enolpyruvate carboxykinase synthesis in the kidney differs markedly from that in the liver.     

Ammonia formation from glutamine isomers by nonacidotic and acidotic perfused rat kidneys.

The following points summarize these findings: (i) there are 2 glutamine utilizing enzyme systems in the rat kidney; (ii) the cytoplasmic glutamyltransferase system hydrolyzes either glutamine isomer while the mitochondrial localized glutaminase 1 is specific for the L-isomer; (iii) the cytoplasmic pathway contributes 70% of the total renal ammonia production in the normal kidney; (iv) chronic metabolic acidosis results in a 20-fold activation of the mitochondrial glutaminase 1 pathway.     

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.     

Inhibition of glutamine synthetase in the mouse kidney: a novel mechanism of adaptation to metabolic acidosis

As part of a study on the regulation of renal ammoniagenesis in the mouse kidney, we investigated the effect of chronic metabolic acidosis on glutamine synthesis by isolated mouse renal proximal tubules. The results obtained reveal that, in tubules from control mice, glutamine synthesis occurred at high rates from glutamate and proline and, to a lesser extent, from ornithine, alanine, and aspartate. A 48 h, metabolic acidosis caused a marked inhibition of glutamine synthesis from near-physiological concentrations of both alanine and proline that were avidly metabolized by the tubules; metabolic acidosis also greatly stimulated glutamine utilization and metabolism. These effects were accompanied by a large increase (i) in alanine, proline, and glutamine gluconeogenesis and (ii) in ammonia accumulation from proline and glutamine. In the renal cortex of acidotic mice, the activity of phosphoenolpyruvate carboxykinase increased 4-fold, but that of glutamate dehydrogenase did not change; in contrast with what is known in the rat renal cortex, metabolic acidosis markedly diminished the glutamine synthetase activity and protein level, but not the glutamine synthetase mRNA level in the mouse renal cortex. These results strongly suggest that, in the mouse kidney, glutamine synthetase is an important regulatory component of the availability of the ammonium ions to be excreted for defending systemic acid-base balance. Furthermore, they show that, in rodents, the regulation of renal glutamine synthetase is species-specific.     

Renal ammoniagenesis in rats made acutely acidotic by swimming

Rats develop metabolic acidosis acutely after exercise by swimming. Renal cortical slices from exercised rats show an increase in both ammoniagenesis and gluconeogenesis from glutamine. In addition, plasma from the exercised rats also stimulates ammoniagenesis in renal cortical slices from normal rats. In exercised rats renal phosphate dependent glutaminase shows a 200% activation when the enzyme activity is measured at subsaturating concentration of glutamine (1 mM) while only an increase of 12% in Vmax is observed. When kidney slices from normal rats are incubated in plasma from exercised rats an activation of phosphate dependent glutaminase is obtained with a 1.0 mM (100%) but not with 20 mM glutamine as substrate. This activation of phosphate dependent glutaminase at subsaturating levels of substrate may indicate a conformational change in PDG effected by a factor present in the plasma of exercised acidotic rats.     

Cellular and molecular basis of increased ammoniagenesis in potassium deprivation

Hypokalemia is associated with increased ammoniagenesis and stimulation of net acid excretion by the kidney in both humans and experimental animals. The molecular mechanisms underlying these effects remain unknown. Toward this end, rats were placed in metabolic cages and fed a control or K(+)-deficient diet (KD) for up to 6 days. Rats subjected to KD showed normal acid-base status and serum electrolytes composition. Interestingly, urinary NH(4)(+) excretion increased significantly and correlated with a parallel decrease in urine K(+) excretion in KD vs. control animals. Molecular studies showed a specific upregulation of the glutamine transporter SN1, which correlated with the upregulation of glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase. These effects occurred as early as day 2 of KD. Rats subjected to a combined KD and 280 mM NH(4)Cl loading (to induce metabolic acidosis) for 2 days showed an additive increase in NH(4)(+) excretion along with an additive increment in the expression levels of ammoniagenic enzymes GA and GDH compared with KD or NH(4)Cl loading alone. The incubation of cultured proximal tubule cells NRK 52E or LLC-PK(1) in low-K(+) medium did not affect NH(4)(+) production and did not alter the expression of SN1, GA, or GDH in NRK cells. These results demonstrate that K(+) deprivation stimulates ammoniagenesis through a coordinated upregulation of glutamine transporter SN1 and ammoniagenesis enzymes. This effect is developed before the onset of hypokalemia. The signaling pathway mediating these events is likely independent of KD-induced intracellular acidosis. Finally, the correlation between increased NH(4)(+) production and decreased K(+) excretion indicate that NH(4)(+) synthesis and transport likely play an important role in renal K(+) conservation during hypokalemia.     

Renal ammonium production--une vue canadienne

The purpose of this review is to examine the factors regulating ammonium production in the kidney and to place these factors in the perspective of acid-base balance. Renal ammonium production and excretion are required to maintain acid-base balance. However, only a portion of renal ammonium production is specifically stimulated by metabolic acidosis. One should examine urinary ammonium excretion at three levels: distribution of ammonium between blood and urine, augmented glutamine metabolism, and an energy constraint due to ATP balance considerations. With respect to the biochemical regulation of acid-base renal ammonium production, an acute stimulation of alpha-ketoglutarate dehydrogenase by a fall in pH seems to be important but this may not be the entire story. In chronic metabolic acidosis augmented glutamine entry into mitochondria (dog) or increased phosphate-dependent glutaminase activity (rat) become critical to support a high flux rate. Metabolic alterations, which diminish the rate of oxidation of alternate fuels, might also be important. The above principles are discussed in the ketoacidosis of fasting, the clinically important situation of high rates of renal ammonium production.     

Hepatic glutaminase flux regulation of glutamine homeostasis. Studies in vivo

The role of hepatic glutaminase flux in regulating plasma glutamine homeostasis was studied in the intact rat. Interorgan glutamine flow during chronic metabolic acidosis was away from the splanchnic bed and to the kidneys. Hindquarter and hepatic glutamine release were the major sources of glutamine removed by the kidneys. Interorgan glutamate flow was from the liver to the hindquarters and kidneys. Chronic metabolic acidosis reduced arterial glutamine concentration 30%. Acute respiratory acidosis (pH 7.12 +/- 0.02) returned arterial glutamine concentration to normal values, increasing and decreasing hepatic glutamine and glutamate release respectively; renal and gut glutamine removal rates were not decreased. Hepatic unidirectional glutamine utilization measured isotopically was decreased 51% by acute acidosis; unidirectional glutamine production was unchanged. The results are consistent with the proposed role of ammonia-activated hepatic glutaminase in the regulation of glutamine homeostasis during acute acidosis.     

The regulation of phosphate-activated glutaminase activity and glutamine metabolism in the streptozotocin-diabetic rat.

The activity of phosphate-activated glutaminase was increased in the kidney, liver and small intestine of rats made diabetic for 6 days with injection of streptozotocin (75 mg/kg body wt.). Insulin prevented this increase in all three tissues. Treatment with NaHCO3, to correct the acidosis that accompanies diabetes, prevented the increase in renal glutaminase activity, but not that in liver or small intestine. Chemically induced acidosis (NH4Cl solution as drinking water) or alkalosis (NaHCO3 solution as drinking water) increased and decreased, respectively, glutaminase activity in the kidney, but were without significant effect on the activity in liver and small intestine. The increase in glutaminase activity in the small intestine during diabetes was due to an overall increase in the size of this organ, and was only detectable when activity was expressed in terms of whole organ, not mucosal scrapings or isolated enterocytes. Prolonged diabetes (40 days) resulted in an even greater increase in the size and glutaminase activity of the small intestine. Despite this marked increase in capacity for glutamine catabolism, arteriovenous-difference measurements showed a complete suppression of plasma glutamine utilization by the small intestine during diabetes, confirming the report by Brosnan, Man, Hall, Colbourne & Brosnan [(1983) Am. J. Physiol. 235, E261-E265].     

Biochemical and histocytochemical studies on response of ammonia-producing enzymes for nh4cl-induced acidosis.

NH4Cl-induced acidosis in rats resulted in renal enlargement and increase in activities of phosphate-dependent glutaminase and glutamic dehydrogenase. The renal enlargement was associated with protein synthesis but not deoxyribonucleic acid synthesis. In control rats histochemical activity of glutamic dehydrogenase was seen dominantly in the proximal straight tubule. In acidotic rats high activity was noted in the proximal convoluted tubule as well as in the proximal straight tubule. By electron microscopy reaction product was in mitochondria. The results suggest that urine ammonia is produced in mitochondria of epithelial cells in the proximal straight tubule in both normal and acidotic rats. Increased enzyme activity in acidotic rats is largely associated with epithelial cells of the proximal convoluted tubule.     

Identification of zeta-crystallin/NADPH:quinone reductase as a renal glutaminase mRNA pH response element-binding protein

Increased renal ammoniagenesis and bicarbonate synthesis from glutamine during chronic metabolic acidosis facilitate the excretion of acids and partially restore normal acid-base balance. This adaptation is sustained, in part, by a cell-specific stabilization of the glutaminase mRNA that leads to an increased synthesis of the mitochondrial glutaminase. A direct repeat of an 8-base AU sequence within the 3'-nontranslated region of the glutaminase mRNA binds a unique protein with high affinity and specificity. Expression of various chimeric mRNAs in LLC-PK(1)-FBPase(+) cells demonstrated that a single 8-base AU sequence is both necessary and sufficient to function as a pH response element (pH RE). A biotinylated oligoribonucleotide containing the direct repeat was used as an affinity ligand to purify the pH RE-binding protein from a cytosolic extract of rat renal cortex. The purified binding activity retained the same specific binding properties as observed with crude extracts and correlated with the elution of a 36-kDa protein. Microsequencing by mass spectroscopy and Western blot analysis were used to identify this protein as zeta-crystallin/NADPH:quinone reductase. The purified protein contained eight tryptic peptides that were identical to sequences found in mouse zeta-crystallin and three peptides that differed by only a single amino acid. The observed differences may represent substitutions found in the rat homolog. A second protein purified by this protocol was identified as T-cell-restricted intracellular antigen-related protein (TIAR). However, the purified TIAR neither bound nor affected the binding of zeta-crystallin/NADPH:quinone reductase to the pH RE. Furthermore, specific antibodies to zeta-crystallin, but not TIAR, blocked the formation of the complex between the pH RE and either the crude cytosolic extract or the purified protein. Thus, zeta-crystallin/NADPH:quinone reductase is a pH response element-binding protein.     

Effect of metabolic or respiratory acidosis on rabbit renal medullary proton-ATPase

Distal urinary acidification is thought to be mediated by an H+-ATPase sensitive to N-ethylmaleimide and dicyclohexyl-carbodiimide. We have studied the effect of chronic metabolic acidosis (NH4Cl for 3 days) or respiratory acidosis (inhalation of 10% CO2 for 2 days) on the H+-ATPase of plasma membranes prepared from the medulla. The enzymatic assay for the H+-ATPase was performed in the presence of ouabain and oligomycin and in the absence of Ca. H+-transport activity was assessed by the quenching of acridine orange in the presence of ATP. The 15-25% sucrose gradient fraction was enriched 40-fold in enzymatic activity over the homogenate, and 8-fold in enzymatic activity and 4-fold in H+-transport activity over the fluffy fraction (38,000 X g). Metabolic acidosis (pH less than 7.31) or chronic hypercapnia (PCO2 greater than 66 mmHg; 1 mmHg = 133.3 Pa) was induced for 2-3 days. Both groups showed the same enrichment factor in enzymatic and H+-transport assays as the control rabbits. Enzymatic and H+-transport activities, however, were not different between animals with respiratory acidosis and controls. Kinetic studies failed to disclose an increase in Vmax (673 vs. 702 mumol/(mg protein.min] or a decrease in Km (0.43 vs. 0.48 mM) in chronic hypercapnia as compared with controls. Metabolic acidosis also failed to increase H+-ATPase activity. These data demonstrate that the H+-ATPase of renal medulla does not display the expected increase in activity during acidosis. The role of this H+-ATPase in the adaptation to acidosis remains to be determined.     

Glutamine metabolism in the kidney during induction of, and recovery from, metabolic acidosis in the rat

Experiments were carried out on rats to evaluate the possible regulatory roles of renal glutaminase activity, mitochondrial permeability to glutamine, phosphoenolpyruvate carboxykinase activity and systemic acid–base changes in the control of renal ammonia (NH₃ plus NH₄⁺) production. Acidosis was induced by drinking NH₄Cl solution ad libitum. A pronounced metabolic acidosis without respiratory compensation [pH=7.25; HCO₃⁻=16.9mequiv./litre; pCO₂=40.7mmHg (5.41kPa)] was evident for the first 2 days, but thereafter acid–base status returned towards normal. This improvement in acid–base status was accompanied by the attainment of maximal rates of ammonia excretion (onset phase) after about 2 days. A steady rate of ammonia excretion was then maintained (plateau phase) until the rats were supplied with tap water in place of the NH₄Cl solution, whereupon pCO₂ and HCO₃⁻ became elevated [55.4mmHg (7.37kPa) and 35.5mequiv./litre] and renal ammonia excretion returned to control values within 1 day (recovery phase). Renal arteriovenous differences for glutamine always paralleled rates of ammonia excretion. Phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase activities and the rate of glutamine metabolism (NH₃ production and O₂ consumption) by isolated kidney mitochondria all increased during the onset phase. The increases in glutaminase and in mitochondrial metabolism continued into the plateau phase, whereas the increase in the carboxykinase reached a plateau at the same time as did ammonia excretion. During the recovery phase a rapid decrease in carboxykinase activity accompanied the decrease in ammonia excretion, whereas glutaminase and mitochondrial glutamine metabolism in vitro remained elevated. The metabolism of glutamine by kidney-cortex slices (ammonia, glutamate and glucose production) paralleled the metabolism of glutamine in vivo during recovery, i.e. it returned to control values. The results indicate that the adaptations in mitochondrial glutamine metabolism must be regulated by extra-mitochondrial factors, since glutamine metabolism in vivo and in slices returns to control values during recovery, whereas the mitochondrial metabolism of glutamine remains elevated.     

Effect of streptozotocin-induced diabetes and insulin treatment on the synthesis of hexokinase II in the skeletal muscle of the rat

The relative rate of synthesis of hexokinase II in the skeletal muscle of the normal, streptozotocin-diabetic, and diabetic insulin-treated rat was determined by the rate of incorporation of [3H]leucine into hexokinase II and the total cytosolic proteins to determine if the rate of hexokinase II synthesis was altered relative to that of the average protein. This relative rate of synthesis of hexokinase II is approximately 1.9 times higher in the normal than in the diabetic rat. The administration of insulin to the diabetic animal increases the rate of hexokinase synthesis to approximately normal levels. An enzyme-linked immunosorbent assay procedure was developed to determine the amount of hexokinase II protein in the skeletal muscle extracts, and immunoprecipitation was utilized to determine the hexokinase II activity. The specific activity of hexokinase II was determined from these analyses. The specific activity of hexokinase II was the same in the skeletal muscle extracts from normal, streptozotocin-diabetic, and diabetic insulin-treated rats. These results suggest that the decrease in muscle hexokinase activity is not caused by the loss of an activator of the enzyme nor by the increased formation of a hexokinase inhibitor in streptozotocin-induced diabetes; rather the decrease in hexokinase II activity reported in diabetic rats relative to normal animals is a result of decreased synthesis coupled to increased degradation in the diabetic relative to the normal animal.