The purine nucleotide cycle and ammoniagenesis in rat kidney tubules

The contribution of the purine nucleotide cycle to renal ammoniagenesis was examined in cortical tubule suspensions prepared from acidotic rats and incubated with [alpha-15N]glutamine, [15N]glutamate, or [15N]aspartate. Labeling of ammonia and adenine nucleotides was determined after enzymatic transformations designed to circumvent the technical problem that 15NH3 and H2O have the same nominal mass. Labeling of the adenine nucleotide was undetectable (less than 10%) even after 1 h of incubation. From the measured concentrations of adenine nucleotides and ammonia and the labeling of the ammonia, the flux through the purine nucleotide cycle was calculated to account for less than 1% of the deamination of alpha-amino groups from all three substrates. The glutamate dehydrogenase reaction is therefore the likely pathway for deamination. The rate of 15NH3 production from [alpha-15N]glutamine was two or three times greater than from added [15N]glutamate, indicating a preference for intracellularly generated glutamate. 15NH3 production from added [15N]aspartate was similar to and perhaps slightly greater than that from added [15N]glutamate.     

The relationship between glutamate deamination and gluconeogenesis in kidney.

The effect of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32], was tested on NH3 formation via the purine nucleotide cycle and glutamate dehydrogenase (EC 1.4.1.2). NH3 excretion in rats increased 70-fold after 48 h of NH4Cl feeding, from 12.2 +/- 4.5 to 862 +/- 190 mumol/mg of creatinine. At 4 h after a single intraperitoneal injection of 3-mercaptopicolinate into NH4Cl-fed rats, NH3 excretion was inhibited by 93%. Kidneys of NH4Cl-fed plus 3-mercaptopicolinate-treated rats, compared with those of NH4Cl-fed rats, showed a 3.5-fold increase in the content of IMP, 5-fold increase in adenylosuccinate, 4-fold increase in aspartate, and a 30% increase in AMP. 3-Mercaptopicolinate completely inhibited NH3 and glucose formation from glutamate in tubules from acidotic rats and NH3 formation from aspartate in kidney perfusion experiments. When transamination in tubules was prevented by 2-amino-4-methoxy-trans-but-3-enoic acid, formation of glucose, but not of NH3, from glutamate was inhibited. 3-Mercaptopicolinate completely inhibited NH3 formation from aspartate in the presence of the aminotransferase inhibitor in kidney tubules. The data show that NH3 can be formed via glutamate dehydrogenase and the purine nucleotide cycle at significant and approximately equal rates. 3-Mercaptopicolinate has no direct effect on NH3 formation via glutamate dehydrogenase, but inhibits that via the purine nucleotide cycle. We conclude that gluconeogenesis is not regulatory for NH3 formation in kidney.     

Effect of 5-amino-4-imidazolecarboxamide riboside on renal ammoniagenesis. Study with [15N]aspartate

[15N]Aspartate and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) were used to evaluate the contribution of the purine nucleotide cycle to ammonia production in renal tubules isolated from control and chronically acidotic rats. Addition of 1 mM AICAriboside to incubation medium containing 2.5 mM [15N] aspartate significantly stimulated production of 15NH3 and 15N in the 6-amino group of adenine nucleotides during a 30-min incubation. In tubules from both control and acidotic animals, the levels of ATP, AMP, and NH3 were increased. In contrast, 5 mM AICAriboside inhibited 15NH3 production and reduced the total purine nucleotide content. In tubules from acidotic rats, enrichment in 15NH3 exceeded that in the 6-amino group of the adenine nucleotides, indicating that no precursor-product relationship existed between the purine nucleotide cycle and ammonia. Conversely, in tubules from control rats, 15N enrichment in the 6-amino group of the adenine nucleotides exceeded that in NH3. This relationship obtained whether or not AICAriboside was included in the incubation mixture. The current investigations show that the metabolism of aspartate through the purine nucleotide cycle is lower in renal tubules obtained from chronically acidotic rats than in control tubules. The observations indicate that AICAriboside has a biphasic effect on renal ammoniagenesis and adenine nucleotide synthesis, and suggest a possible clinical use of AICAriboside in cases of impaired ammonia formation in renal failure.     

The purine nucleotide cycle and ammonia formation from glutamine by rat kidney slices.

To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.     

Metabolism of glutamine and glutamate by rat renal tubules. Study with 15N and gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry was utilized to study the metabolism of [15N]glutamate, [2-15N]glutamine, and [5-15N]glutamine in isolated renal tubules prepared from control and chronically acidotic rats. The main purpose was to determine the nitrogen sources utilized by the kidney in various acid-base states for ammoniagenesis. Incubations were performed in the presence of 2.5 mM 15N-labeled glutamine or glutamate. Experiments with [5-15N]glutamine showed that in control animals approximately 90% of ammonia nitrogen was derived from 5-N of glutamine versus 60% in renal tubules from acidotic rats. Experiments with [2-15N]glutamine or [15N]glutamate indicated that in chronic acidosis approximately 30% of ammonia nitrogen was derived either from 2-N of glutamine or glutamate-N by the activity of glutamate dehydrogenase. Flux through glutamate dehydrogenase was 6-fold higher in chronic acidosis versus control. No 15NH3 could be detected in renal tubules from control rats when [2-15N]glutamine was the substrate. The rates of 15N transfer to other amino acids and to the 6-amino groups of the adenine nucleotides were significantly higher in normal renal tubules versus those from chronically acidotic rats. In tubules from chronically acidotic rats, 15N abundance in 15NH3 and the rate of 15NH3 appearance were significantly higher than that of either the 6-amino group of adenine nucleotides or the 15N-amino acids studied. The data indicate that glutamate dehydrogenase activity rather than glutamate transamination is primarily responsible for augmented ammoniagenesis in chronic acidosis. The contribution of the purine nucleotide cycle to ammonia formation appears to be unimportant in renal tubules from chronically acidotic rats.     

Sources of ammonia for mammalian urea synthesis.

The initial rate of incorporation of [15N]alanine into the 6-amino group of the adenine nucleotides in rat hepatocytes was about one-eighteenth of the rate of incorporation into urea. Thus the purine nucleotide cycle cannot provide most of the ammonia needed in urea synthesis for the carbamoyl phosphate synthase reaction (EC 2.7.2.5). On the other hand, contrary to the view expressed by McGivan & Chappell [(1975) FEBS Lett. 52, 1--7], the experiments support the view that hepatic glutamate dehydrogenase can supply the required ammonia.     

Relative role of the glutaminase, glutamate dehydrogenase, and AMP-deaminase pathways in hepatic ureagenesis: studies with 15N.

We have studied the relative roles of the glutaminase versus glutamate dehydrogenase (GLDH) and purine nucleotide cycle (PNC) pathways in furnishing ammonia for urea synthesis. Isolated rat hepatocytes were incubated at pH 7.4 and 37 degrees C in Krebs buffer supplemented with 0.1 mM L-ornithine and 1 mM [2-15N]glutamine, [5-15N]glutamine, [15N]aspartate, or [15N]glutamate as the sole labeled nitrogen source in the presence and absence of 1 mM amino-oxyacetate (AOA). A separate series of incubations was carried out in a medium containing either 15N-labeled precursor together with an additional 19 unlabeled amino acids at concentrations similar to those of rat plasma. GC-MS was utilized to determine the precursor product relationship and the flux of 15N-labeled substrate toward 15NH3, the 6-amino group of adenine nucleotides ([6-15NH2]adenine), 15N-amino acids, and [15N]urea. Following 40 min incubation with [15N]aspartate the isotopic enrichment of singly and doubly labeled urea was 70 and 20 atom % excess, respectively; with [15N]glutamate these values were approximately 65 and approximately 30 atom % excess for singly and doubly labeled urea, respectively. In experiments with [15N]aspartate as a sole substrate 15NH3 enrichment exceeded that in [6-NH2]adenine, indicating that [6-15NH2]adenine could not be a major precursor to 15NH3. Addition of AOA inhibited the formation of [15N]glutamate, 15NH3 and doubly labeled urea from [15N]aspartate. However, AOA had little effect on [6-15NH2]adenine production. In experiments with [15N]glutamate, AOA inhibited the formation of [15N]aspartate and doubly labeled urea, whereas 15NH3 formation was increased. In the presence of a physiologic amino acid mixture, [15N]glutamate contributed less than 5% to urea-N. In contrast, the amide and the amino nitrogen of glutamine contributed approximately 65% of total urea-N regardless of the incubation medium. The current data indicate that when glutamate is a sole substrate the flux through GLDH is more prominent in furnishing NH3 for urea synthesis than the flux through the PNC. However, in experiments with medium containing a mixture of amino acids utilized by the rat liver in vivo, the fraction of NH3 derived via GLDH or PNC was negligible compared with the amount of ammonia derived via the glutaminase pathway. Therefore, the current data suggest that ammonia derived from 5-N of glutamine via glutaminase is the major source of nitrogen for hepatic urea-genesis.     

The mechanism of ammonia production and the effect of mechanical work load on proteolysis and amino acid catabolism in isolated perfused rat heart.

The effect of mechanical work load on net proteolysis, amino acid catabolism and ammonia production was studied in isolated perfused beating or K+-arrested hearts. Net proteolysis was about 16 mumol/g dry wt. during 1h and was not affected by the mechanical work. The combined catabolic rate of the major amino acids was 7.1 mumol/g dry wt. in the beating heart and 2.1 mumol/g dry wt. in the arrested heart during the 1 h experimental period. The main differences lay in the deamination of aspartate plus glutamate, which was inhibited by 60% during low energy consumption, and in net alanine synthesis, which was increased by 94%. The ammonia production plus its conversion into amide nitrogen was 9.2 and 3.4 mumol/g dry wt. in the beating and arrested heart respectively during 1 h. The decrease in the total adenine nucleotide pool during the 1 h perfusion was very low, 1.0 and 0.5 mumol/g dry wt. in the beating and arrested hearts respectively, and did not contribute significantly to ammonia production. Thus ammonia production is dependent on the cellular energy state, whereas net proteolysis is not. The maximal capacities of the purine nucleotide cycle and the glutamate dehydrogenase reaction towards deamination were much higher than the observed ammonia-production rates. The anaplerotic role of amino acid catabolism in the myocardium is discussed.     

Effects of ischaemia on metabolite concentrations in rat liver

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.     

Utilization of [15N]glutamate by cultured astrocytes.

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.     

Effect of 5-amino-4-imidazolecarboxamide riboside (AICA-riboside) on the purine nucleotide synthesis and growth of rat kidney cells in culture: study with [15N]aspartate

The present investigation evaluates the effect of AICA-Riboside on the synthesis of purine nucleotides and the growth of normal rat kidney cells in culture. Experiments in the presence and absence of various concentrations of AICA-Riboside were conducted with Dulbecco's Modified Eagle's Medium supplemented with either 1 mM [15N]aspartate or [14N]aspartate. Addition of 50 microM AICA-Riboside to the incubation medium significantly stimulated intracellular adenine nucleotide concentrations following incubation for 48 hours. This stimulation was associated with augmented cell growth and DNA concentration. In contrast, with concentrations above 100 microM of AICA-Riboside in the incubation medium, there was a remarkable inhibition of cell growth and a significant depletion of intracellular pools of adenine nucleotides and DNA. Experiments with [15N]aspartate showed that the initial rate (0-24 hours) of [6-15NH2]adenine nucleotide formation from 1 mM [15N]aspartate was 38.8 +/- 9.6, 67.9 +/- 12.5, and 20.1 +/- 3.8 pmol h-1/10(6) cells in the presence of 0 (control), 50 microM and 500 microM AICA-Riboside, respectively. These observations indicate that the main effect of AICA-Riboside is on the formation of AMP from aspartate and IMP via the sequential action of adenylosuccinate synthetase and adenylosuccinate lyase. The current studies suggest that AICA-Riboside could be used as a factor mediating renal cell mitosis in culture. AICA-Riboside has a biphasic effect on the growth of renal epithelial cells in culture and on their intracellular purine nucleotides and DNA concentration.     

Formation of the intermediate products of the tricarboxylic acid cycle and ammonia from free amino acids in anoxic heart muscle

Glutamate and aspartate showed the highest rate of catabolism in oxygenated isolated rat heart with the formation of glutamine, asparagine and alanine. Under anoxia, the catabolism of branch chained amino acids and that of lysine, proline, arginine and methionine was inhibited. However, glutamate and aspartate catabolized at a higher rate as compared with oxygenation. Alanine was the product of their excessive degradation. During oxygenation, 70% of ammonia were produced via deamination of amino acids. Under anaerobic conditions the participation of amino acids in ammoniagenesis decreased to 4%; the principal source of ammonia was the adenine nucleotide pool. The total pool of the tricarboxylic acid cycle intermediates increased 2.5-fold due to accumulation of succinate. The data obtained suggest that the constant influx of intermediates into the cycle from amino acids is supported by coupled transamination of glutamate and aspartate. This leads to the formation of ATP and GTP in the tricarboxylic acid cycle during blocking of aerobic energy production.     

Ammonia and amino acid metabolism in human skeletal muscle during exercise.

This review focuses on the ammonia and amino acid metabolic responses of active human skeletal muscle, with a particular emphasis on steady-state exercise. Ammonia production in skeletal muscle involves the purine nucleotide cycle and the amino acids glutamate, glutamine, and alanine and probably also includes the branched chain amino acids as well as aspartate. Ammonia production is greatest during prolonged, steady state exercise that requires 60-80% VO2max and is associated with glutamine and alanine metabolism. Under these circumstances it is unresolved whether the purine nucleotide cycle (AMP deamination) is active; if so, it must be cycling with no IMP accumulation. It is proposed that under these circumstances the ammonia is produced from slow twitch fibers by the deamination of the branched chain amino acids. The ammonia response can be suppressed by increasing the carbohydrate availability and this may be mediated by altering the availability of the branched chain amino acids. The fate of the ammonia released into the circulation is unresolved, but there is indirect evidence that a considerable portion may be excreted by the lung in expired air.     

The effects of increased heart work on the tricarboxylate cycle and its interactions with glycolysis in the perfused rat heart

1. The work of the perfused rat heart was acutely increased by raising the aortic pressure in the Langendorff preparation from 50 to 120mmHg; within 1 min in perfusions with media containing glucose or glucose+acetate, rates of oxygen consumption and tricarboxylate-cycle turnover increased 2.5-fold, glycolysis rate doubled and oxidation of triglyceride fatty acid was strikingly enhanced. 2. Increased cardiac work had no significant effects on the heart concentrations of creatine phosphate, ATP, ADP or 5′-AMP. The only significant changes in tricarboxylate-cycle intermediates were a decrease in malate in perfusions with glucose and decreases in acetyl-CoA and citrate and an increase in aspartate in perfusions with glucose+acetate. 3. Measurements of intracellular concentrations of hexose phosphates, glucose and glycogen indicated that work accelerated glycolysis by activation of phosphofructokinase and subsequently hexokinase; the activation could not be accounted for by changes in the known effectors of phosphofructokinase. 4. Acetate at either perfusion pressure increased heart concentrations of acetyl-CoA, citrate, glutamate and malate and decreased that of aspartate; acetate increased tricarboxylate-cycle turnover by 50–60% and inhibited glycolysis and pyruvate oxidation. 5. In view of the markedly different effects of acetate and of cardiac work on the concentrations of cycle intermediates the changes that accompany acetate utilization may be specifically concerned with the regulatory functions of the cycle in control of glycolysis and pyruvate oxidation and not with the associated increase in cycle turnover. It is suggested that the concentrations of key metabolites controlling the rate of cycle turnover may fluctuate with each heart beat and that this may explain why no significant changes (for example, in adenine nucleotide concentrations) have been detected with increased work in the present study.     

Cerebral Aspartate Utilization: Near-Equilibrium Relationships in Aspartate Aminotransferase Reaction

Abstract: The pathways of nitrogen transfer from 50 μM [¹⁵N]aspartate were studied in rat brain synaptosomes and cultured primary rat astrocytes by using gas chromatography-mass spectrometry technique. Aspartate was taken up rapidly by both preparations, but the rates of transport were faster in astrocytes than in synaptosomes. In synaptosomes, ¹⁵N was incorporated predominantly into glutamate, whereas in glial cells, glutamine and other ¹⁵N-amino acids were also produced. In both preparations, the initial rate of N transfer from aspartate to glutamate was within a factor of 2-3 of that in the opposite direction. The rates of transamination were greater in synaptosomes than in astrocytes. Omission of glucose increased the formation of [¹⁵N]-glutamate in synaptosomes, but not in astrocytes. Rotenone substantially decreased the rate of transamination. There was no detectable incorporation of ¹⁵N from labeled aspartate to 6-amino-¹⁵N-labeled adenine nucleotides during 60-min incubation of synaptosomes under a variety of conditions; however, such activity could be demonstrated in glial cells. The formation of ¹⁵N-labeled adenine nucleotides was marginally increased by the presence of 1 mM aminooxyacetate, but was unaffected by pretreatment with 1 mM 5-amino-4-imidazolecarboxamide ribose. It is concluded that (1) aspartate aminotransferase is near equilibrium in both synaptosomes and astrocytes under cellular conditions, but the rates of transamination are faster in the nerve endings; (2) in the absence of glucose, use of amino acids for the purpose of energy production increases in synaptosomes, but may not do so in glial cells because the latter possess larger glycogen stores; and (3) nerve endings have a very limited capacity for salvage of the adenine nucleotides via the purine nucleotide cycle.     

Purine nucleotide cycle. Evidence for the occurrence of the cycle in brain.

Cell-free extracts of rat brain catalyze the reactions of the purine nucleotide cycle. Ammonia is formed during the deamination but not the amination phase of the cycle. The activity of adenylate deaminase in brain is sufficient to account for the maximum rates of ammonia production that have been reported. The activity of glutamate dehydrogenase is not sufficient to account for these rates of ammonia production. The activities of adenylosuccinate synthetase and adenylosuccinase are nearly sufficient to account for the steady state rates of ammonia production observed in brain. Demonstration of the cycle in extracts of brain is complicated by the occurrence of side reactions, in particular those catalyzed by phosphomonoesterase, nucleoside phosphorylase, and guanase.     

Fuel utilization in colonocytes of the rat.

In incubated colonocytes isolated from rat colons, the rates of utilization O2, glucose or glutamine were linear with respect to time for over 30 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 30 min. Glutamine, n-butyrate or ketone bodies were the only substrates that caused increases in O2 consumption by isolated incubated colonocytes. The maximum activity of hexokinase in colonic mucosa is similar to that of 6-phosphofructokinase. Starvation of the donor animal decreased the activities of hexokinase and 6-phosphofructokinase, whereas it increased those of glucose-6-phosphatase and fructose-bisphosphatase. Isolated incubated colonocytes utilized glucose at about 6.8 mumol/min per g dry wt., with lactate accounting for 83% of glucose removed. These rates were not affected by the addition of glutamine, acetoacetate or n-butyrate, and starvation of the donor animal. Isolated incubated colonocytes utilized glutamine at about 5.5 mumol/min per g dry wt., which is about 21% of the maximum activity of glutaminase. The major end-products of glutamine metabolism were glutamate, aspartate, alanine and ammonia. Starvation of the donor animal decreased the rate of glutamine utilization by colonocytes, which is accompanied by a decrease in glutamate formation and in the maximum activity of glutaminase. Isolated incubated colonocytes utilized acetoacetate at about 3.5 mumol/min per g dry wt. This rate was not markedly affected by addition of glucose or by starvation of the donor animal. When colonocytes were incubated with n-butyrate, both acetoacetate and 3-hydroxybutyrate were formed, with the latter accounting for only about 19% of total ketones produced.     

The purine nucleotide cycle inHelix hepatopancreas

Summary The enzymes adenylosuccinate synthetase (EC 6.3.4.4 IMP: L-aspartate ligase [GDP-forming]), adenylosuccinate lyase (EC 4.3.2.2) and AMP deaminase (EC 3.5.4.6 AMP aminohydrolase) were demonstrated inHelix aspersa hepatopancreas tissue. The presence of these enzymes along with high levels of aspartate transaminase is presumptive evidence for the operation in this tissue of the purine nucleotide cycle. In the absence of evidence that glutamate dehydrogenase acts to release ammonia during amino acid catabolism, it is suggested that the purine nucleotide cycle serves this function. Glutamine synthetase (EC 6.3.1.2 L-glutamate: ammonia ligase [ADP-forming]) was shown to be present primarily in the cytosolic fraction ofHelix hepatopancreas. Since the operation of the purine nucleotide cycle results in the release of ammonia in the cytosol, the localization of glutamine synthetase in this compartment indicates that it is the primary ammonia-detoxifying enzyme and is consistent with the suggestion that the purine nucleotide cycle serves as the major pathway for amino acid catabolism.Supported by grants from the USPHS National Institute of Allergic and Infections Diseases (AI 05006) and the National Science Foundation (PCM-75-13161)     

Inorganic nitrogen assimilation in yeasts: alteration in enzyme activities associated with changes in cultural conditions and growth phase

Ammonia assimilation has been investigated in four strains of Saccharomyces cerevisiae by measuring, at intervals throughout the growth cycle, the activities of several enzymes concerned with inorganic ammonia assimilation. Enzyme activities in extracts of cells were compared after growth in complete and defined media. The effect of shift from growth in a complete to growth in a defined medium (and the reverse) was also determined. The absence of aspartase (EC 4.3.1.1, l-aspartate-ammonia lyase) activity, the low specific activities of alanine dehydrogenase, glutamine synthetase [EC 6.3.1.2, l-glutamate-ammonia ligase (ADP)], and the marked increase in activity of the nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase (NADP-GDH) [EC 1.4.1.4, l-glutamate:NADP-oxidoreductase (deaminating)] during the early stages of growth support the conclusion that yeasts assimilate ammonia primarily via glutamate. The NADP-GDH showed a rapid increase in activity just before the initiation of exponential growth, reached a maximum at the mid-exponential stage, and then gradually declined in activity in the stationary phase. The NADP-GDH reached a higher level of activity when the yeasts were grown on the defined medium as compared with complete medium. The nicotinamide adenine dinucleotide-linked glutamate dehydrogenase (NAD-GDH) [EC 1.4.1.2, l-glutamate:NAD-oxidoreductase (deaminating)] showed only slight increases in activity during the exponential phase of growth. There was an inverse relationship in that the NADP-GDH increased in activity as the NAD-GDH decreased. The NAD-GDH activity was higher after growth on the complete medium. The glutamate-oxaloacetate transaminase (EC 2.6.1.1. l-aspartate:2-oxoglutarate aminotransferase) activity rose and fell in parallel with the NADP-GDH, although its specific activity was somewhat lower. Although other ammonia-assimilatory enzymes were demonstrable, it seems unlikely that their combined activities could account for the remainder of the ammonia-assimilatory capacity not accounted for by the NADP-GDH. The ability of aspartate to serve as effectively as glutamate as the sole source of nitrogen for the growth of yeast apparently resides in their ability to utilize aspartate for amino acid biosynthesis via transamination.     

[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry.

The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.