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

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

beta-2-Aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. A new activator of glutaminase in intact rat liver mitochondria

beta-(+/-)-2-Aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (BCH) stimulated, in a concentration-dependent manner, the formation of glutamate by mitochondria isolated from rat liver and incubated with 20 mM glutamine. Maximum enhancement was seen with 10 mM BCH while 5 mM leucine was without effect. The initial lag in the rate of glutamate formation was not eliminated by BCH. Preincubation of the mitochondria without glutamine also did not abolish the lag period; to the contrary, it resulted in a progressive deactivation of the glutaminase. The decrease in enzyme activity during the preincubation without glutamine was partially reversed by the addition of either 10 mM BCH or 1.4 mM NH4Cl and was essentially abolished by their combined action. The apparently sigmoid rise in the activity of glutaminase with increasing concentration of glutamine became hyperbolic in the presence of 1.4 mM NH4Cl. BCH stimulated the NH4Cl-activated glutaminase in the entire range of glutamine concentrations studied (2-40 mM) without changing the S50 value. In mitochondria disrupted by repeated cycles of freezing and thawing, the enzymatic activity was maximal even in the absence of BCH. It is postulated that BCH is a potent activator of mitochondrial glutaminase and that manifestation of its action requires intact organelle structure. In addition, it is concluded that BCH-induced stimulation of glutamine catabolism in isolated hepatocytes (Zaleski, J., Wilson, D. F., and Erecinska, M. (1986) J. Biol. Chem. 261, 14082-14090) is the consequence of activation of the mitochondrial glutaminase.     

The stimulation of glutamine hydrolysis in isolated rat liver mitochondria by Mg2+ depletion and hypo-osmotic incubation conditions.

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.     

Glutamine synthetase and glutaminase activities in various hepatoma cells

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.     

Inhibition of CA V decreases glucose synthesis from pyruvate

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.     

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

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

Origin of the ammonia used for mitochondrial citrulline synthesis as revealed by 13C-15N spin coupling patterns observed by 13C NMR.

The sources of ammonia used by isolated, intact rat liver mitochondria in the production of citrulline have been investigated in situ using a novel methodology based on the analysis of 13C-15N heteronuclear couplings observed by 13C NMR. Isolated mitochondria from rat liver were incubated with ornithine, 13CO3H- and 15NH4Cl, using unlabeled glutamate or glutamine as alternative, intramitochondrial nitrogen donors. The production of (7-13C, 8-15N) or (7-13C, 8-14N) citrulline was determined in situ by 13C NMR and the relative proportions of 15N- and 14N-citrullines confirmed by high resolution 13C NMR analysis of the C-7 citrulline resonance observed in perchloric acid extracts prepared at the end of the incubations. The 15N fractional enrichment of the intramitochondrial NH3 pool was manipulated either by modifying the 15N enrichment of added 15NH4Cl, or by altering the concentration of the unlabeled nitrogen donors in the incubation medium. Fractional 15N enrichments measured in the N-8 nitrogen of the resulting (7-13C) citrulline closely paralleled those of the external 15NH4Cl with minor dilutions derived from the unlabeled nitrogen contribution from the alternative substrates. In the presence of 10 mM 15NH4Cl, 10 mM glutamate contributed 4% of the citrulline N-8 nitrogen. Under similar conditions, the contribution of nitrogen from 10 mM glutamine to N-8 citrulline was 6%. These results indicate that the primary source of ammonia used for citrulline synthesis by isolated, intact rat liver mitochondria is extramitochondrial, providing also an illustration of the use of 13C-15N spin coupling patterns observed by 13C NMR, as a new tool in the study of ammonia metabolism.     

Effect of l-leucine on the nitrogen metabolism of isolated rat liver mitochondria

1. l-Leucine strongly activated intramitochondrial glutamate dehydrogenase in the direction of glutamate synthesis. 2. In the deamination direction, the enzyme was not stimulated by leucine. This was probably due to a rate-limiting transport of glutamate across the mitochondrial membrane. 3. The effect of leucine on the kinetic constants of glutamate dehydrogenase in a mitochondrial sonicate was studied. 4. In isolated mitochondria, leucine did not stimulate the synthesis of citrulline with glutamate as the source of NH(3). 5. Leucine very markedly stimulated the synthesis of glutamate from added 2-oxoglutarate+NH(4)Cl. 6. Under conditions where glutamate and citrulline could be synthesized simultaneously from added NH(4)Cl, leucine greatly increased glutamate synthesis at the expense of citrulline synthesis. 7. It is suggested that the intramitochondrial leucine concentration may be a factor influencing the nitrogen metabolism of the liver cell.     

Mitochondrial citrulline synthesis from ammonia and glutamine in the liver of ureogenic air-breathing catfish, Clarias batrachus (Linnaeus)

The possible synthesis of citrulline, a rate limiting step for urea synthesis via the ornithine-urea cycle (OUC) in teleosts was tested both in the presence of ammonia and glutamine as nitrogen-donating substrates by the isolated liver mitochondria of ureogenic air-breathing walking catfish, C. batrachus. Both ammonia and glutamine could be used as nitrogen-donating substrates for the synthesis of citrulline by the isolated liver mitochondria, since the rate of citrulline synthesis was almost equal in presence of both the substrates. The citrulline synthesis by the isolated liver mitochondria requires succinate at a concentration of 0.1 mM as an energy source, and also requires the involvement of intramitochondrial carbonic anhydrase activity for supplying HCO3 as another substrate for citrulline synthesis. The rate of citrulline synthesis was further stimulated significantly by the isolated liver mitochondria of the fish after pre-exposure to 25 mM NH4Cl for 7 days. Due to possessing this biochemical adaptational strategy leading to the amelioration of ammonia toxicity mainly by channeling ammonia directly and/or via the formation of glutamine to the OUC, this air-breathing catfish could succeed in surviving in high external ammonia, which it faces in its natural habitat in certain seasons of the year.     

Interactions between glutamine metabolism and cell-volume regulation in perfused rat liver

1. In the presence of near-physiological glutamine concentrations, exposure of perfused rat liver to hypotonic perfusion media switched glutamine balance across the liver from net release to net uptake. This was due to both stimulation of flux through glutaminase and inhibition of flux through glutamine synthetase. Conversely, during exposure to hypertonic media, net glutamine release from the liver increased due to inhibition of glutaminase flux and slight stimulation of flux through glutamine synthetase. The effect of perfusate osmolarity on glutaminase flux was observed at an NH4Cl concentration (0.5 mM) sufficient for near-maximal ammonia stimulation of glutaminase. This indicates the involvement of different mechanisms of glutaminase flux control by extracellular osmolarity changes and ammonia. The effects of anisotonicity on flux through glutamine-metabolizing enzymes were fully reversible. Glutamine (0.6 mM) stimulated urea synthesis from NH4Cl (0.5 mM) during hypotonic and normotonic conditions. 2. Exposure to hypotonic and hypertonic media led, after initial liver-cell swelling and shrinkage, respectively to volume-regulatory K+ fluxes which largely restored the initial liver-cell volume despite the continuing osmotic challenge. Even after completion of cell-volume regulatory K+ fluxes, the effects of perfusate osmolarity on hepatic glutamine metabolism persisted. This indicates that in anisotonicity the liver cell is left in an altered metabolic state, even after completion of volume-regulatory responses. 3. During perfusion with isotonic media, addition of glutamine (3 mM) led to an increase of liver mass by about 4% within 2 min, which was accompanied by a net K+ uptake by the liver. Thereafter, the new steady state of increased liver mass was maintained throughout glutamine infusion. When the liver mass had reached this new steady state, a net release of K+ from the liver of about 3 mumol/g liver was observed during the following 10 min. Withdrawal of glutamine was followed by a slow reuptake of K+ and the liver mass returned to its initial value. Following exposure to glutamine (3 mM), the intracellular glutamine concentration (as calculated from glutamine tissue levels, taking into account the extracellular space determined with the [3H]inulin technique) rose from about 1 mM to 30-35 mM within about 12 min, indicating a 10-12-fold concentrative uptake of glutamine into the liver cells and an osmotic challenge for the hepatocyte. When intracellular glutamine had reached its steady-state concentration, net K+ efflux from the liver was also terminated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interconversion of two distinct states of active CF0-CF1 (chloroplast ATPase complex) in chloroplasts

Chloroplast ATPase complex is activated by illumination in the presence or absence of dithiothreitol. ATPase complex which has been activated without dithiothreitol catalyzes ATP hydrolysis which is insensitive to stimulation by NH4Cl and is highly sensitive to medium pH. Addition of dithiothreitol during illumination results in an increase in the stimulating effect of NH4Cl on ATP hydrolysis and a decrease in pH sensitivity of ATP hydrolysis. With increasing time in the dark, the ability of NH4Cl to stimulate ATP hydrolysis decreases and the effect of pH on the ATP hydrolysis increases. The onset of resistance to NH4Cl stimulation and the increase in sensitivity to pH are accelerated by ADP and the acceleration is inhibited by Pi. ATP hydrolysis restores NH4Cl sensitivity and renders the activity more resistant to pH. These results suggest that active chloroplast ATPase complex converts its state reversibly from the NH4Cl-insensitive and highly pH-sensitive one to the NH4Cl-sensitive and relatively pH-insensitive one. The conversion from the former to the latter requires both sulfhydryl compound and energy.     

Adaptive changes in the capacity of systems used for the synthesis of citrulline in rat liver mitochondria in response to high- and low-protein diets

1. Citrulline synthesis was measured in mitochondria from rats fed on a standard diet, a high-protein diet, or on glucose. 2. With NH(4)Cl as the nitrogen source the rate of citrulline synthesis was higher in mitochondria from rats fed on a high-protein diet than in those from rats fed on a standard diet. When rats were fed solely on glucose the rate of synthesis of citrulline from NH(4)Cl was very low. 3. With glutamate as the nitrogen source the relative rates of citrulline synthesis were much lower than when NH(4)Cl was present, but similar adaptive changes occurred. 4. The activity of the mitochondrial glutamate-transporting system increased two to three times on feeding rats on a high-protein diet, but the K(m) for glutamate was unchanged. 5. Adaptive changes in certain intramitochondrial enzymes were also measured. 6. The results were interpreted to indicate that when an excess of substrate was present, citrulline synthesis from NH(4)Cl was rate-limited by the intramitochondrial concentration of N-acetyl-glutamate, but citrulline synthesis from glutamate was rate-limited primarily by the activity of the glutamate-transporting system.     

The intramitochondrial location of the glutaminase isoenzymes of pig kidney

1. The glutaminase activity of pig kidney is located almost entirely in the cortex. 2. Pig renal cortex contains two glutaminases, one phosphate-dependent and one phosphate-independent. Both isoenzymes are localized exclusively in the mitochondria. 3. After sonication of the mitochondria, the phosphate-dependent isoenzyme is entirely soluble, whereas approximately half the phosphate-independent isoenzyme is associated with the membranes. 4. In intact mitochondria, the activities of both isoenzymes respond to changes in the pH of the intramitochondrial compartment. 5. It is concluded that both glutaminase isoenzymes are situated in the intramitochondrial compartment, and that the phosphate-independent glutaminase may be bound to the inside of the inner mitochondrial membrane.     

The stimulatory effect of alloxan diabetes on citrulline formation in rabbit liver mitochondria

The effect of alloxan diabetes on citrulline formation from NH₄Cl and bicarbonate was studied in rabbit liver mitochondria incubated with glutamate or succinate as respiratory substrate, as well as with exogenous ATP in the presence of uncoupler and oligomycin. In contrast to ornithine transcarbamoylase, the activity of carbamoyl-phosphate synthetase (ammonia) was higher in mitochondria from diabetic animals than in those from normal ones. In diabetic rabbits the rates of citrulline synthesis were stimulated under all conditions studied. In contrast, levels of N-acetyglutamate, an activator of carbamoyl-phosphate synthetase (ammonia), were significantly increased only in the presence of glutamate, while the highest rates of citrulline formation occurred in uncoupled mitochondria incubated with exogenous ATP as energy source. Treatment of animals with alloxan resulted in an increase of both the intramitochondiral ATP level and the rate of adenine nucleotide translocation across the mitochondrial membrane. The results indicate that the stimulation of citrulline formation in liver mitochondria of diabetic rabbits is mainly due to an increase in carbamoyl-phosphate synthetase (ammonia) activity and an elevation of content of intramitochondrial ATP, a substrate of this enzyme.     

Crystal structures of CTP synthetase reveal ATP, UTP, and glutamine binding sites

CTP synthetase (CTPs) catalyzes the last step in CTP biosynthesis, in which ammonia generated at the glutaminase domain reacts with the ATP-phosphorylated UTP at the synthetase domain to give CTP. Glutamine hydrolysis is active in the presence of ATP and UTP and is stimulated by the addition of GTP. We report the crystal structures of Thermus thermophilus HB8 CTPs alone, CTPs with 3SO4(2-), and CTPs with glutamine. The enzyme is folded into a homotetramer with a cross-shaped structure. Based on the binding mode of sulfate anions to the synthetase site, ATP and UTP are computer modeled into CTPs with a geometry favorable for the reaction. Glutamine bound to the glutaminase domain is situated next to the triad of Glu-His-Cys as a catalyst and a water molecule. Structural information provides an insight into the conformational changes associated with the binding of ATP and UTP and the formation of the GTP binding site.     

Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined

The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.     

The regulation of carbamoyl phosphate synthase activity in rat liver mitochondria.

The rate at which isolated rat liver mitochondria synthesized citrulline with NH4C1 as nitrogen source was markedly dependent on the protein content of the diet. 2. Citrulline synthesis was not rate-limited by substrate concentration, substrate transport or ornithine transcarbamoylase activity under the conditions used. 3. The intramitochondrial content of an activator of carbamoyl phosphate synthase, assumed to be N-acetyl-glutamate, varied markedly with dietary protein content. The variation in the concentration of this activator was sufficient to account for the observed variation in the rates of citrulline synthesis if this synthesis were rate-limited by the activity of carbamoyl phosphate synthase. 4. The rates of urea formation from NH4Cl as nitrogen source in isolated liver cells showed variations in response to diet that closely paralleled the variations in the rates of citrulline synthesis observed in isolated mitochondria. 5. These results are consistent with the postulate that when NH4Cl plus ornithine are present in an excess, the rate of urea synthesis is regulated at the level of carbamoyl phosphate synthase activity.     

Effects of ammonia and norvaline on lactate metabolism by hepatocytes from starved rats. The use of 14C-labelled lactate in studies of hepatic gluconeogenesis

1. Hepatocytes from starved rats were incubated with l-lactate and NH(4)Cl or norvaline, and the rates of the tricarboxylic acid cycle and of gluconeogenesis were calculated from changes in metabolite concentrations or from radioisotopic data from incubations with labelled lactate or propionate. 2. Gluconeogenesis was stimulated by the addition of 10mm-NH(4)Cl, 5mm-norvaline or 1mm-oleate by 27, 45 and 59% respectively. NH(4)Cl or norvaline also increased lactate uptake. Norvaline inhibited urea synthesis from NH(4)Cl by 85%. 3. The effects of NH(4)Cl and norvaline were not additive. However, NH(4)Cl inhibited and norvaline was without effect on gluconeogenesis from pyruvate, indicating that the two compounds act by different mechanisms. 4. The tricarboxylic acid-cycle flux was increased 80% by lactate, and NH(4)Cl caused a further 25% stimulation. Norvaline had no effect on the tricarboxylic acid-cycle flux. NH(4)Cl and norvaline tripled and doubled, respectively, flux through pyruvate dehydrogenase. 5. Total ATP formation was calculated to range from 470 to 830mumol/h per 100mg of protein, of which the basic metabolic activity accounted for 400-450mumol/h per 100mg of protein. ATP formation does not seem to be rate-limiting for gluconeogenesis. 6. Pyruvate recycling was estimated from the (14)C yield from [1-(14)C]propionate in lactate and glucose to be 10-30% of the flux of phosphoenolpyruvate to glucose. The further addition of NH(4)Cl more than doubled the recycling of pyruvate. 7. [1,4-(14)C]Succinate was rapidly metabolized by hepatocytes. About 20% of the radioactivity was recovered in glucose, indicating that succinate is also metabolized by intact (non-damaged) hepatocytes. 8. It is concluded that the metabolism of lactate by the liver is too complex to allow simple rate measurements with labelled compounds.     

Modulation of gill Na+,K+-ATPase activity by ammonium ions: Putative coupling of nitrogen excretion and ion uptake in the freshwater shrimp Macrobrachium olfersii

The effect of NH4+ ions on (Na+,K+)-ATPase hydrolytic activity was examined in a gill microsomal fraction from M. olfersii. In the absence of NH4+ ions, K+ ions stimulated ATP hydrolysis, exhibiting cooperative kinetics (nH=0.8), to a maximal specific activity of V=556.1+/-22.2 nmol.min(-1).mg(-1) with K(0.5)=2.4+/-0.1 mmol.L(-1). No further stimulation by K+ ions was observed in the presence of 50 mmol.L(-1) NH4+ ions. ATP hydrolysis was also stimulated by NH4+ ions obeying Michaelian kinetics to a maximal specific activity of V=744.8+/-22.3 nmol.min(-1).mg(-1) and KM=8.4+/-0.2 mmol.L(-1). In the presence of 10 mmol.L(-1) K+ ions, ATP hydrolysis was synergistically stimulated by NH4+ ions to V=689.8+/-13.8 nmol.min(-1).mg(-1) and K(0.5)=6.6+/-0.1 mmol.L(-1), suggesting that NH4+ ions bind to different sites than K+ ions. PNPP hydrolysis was also stimulated cooperatively by K+ or NH4+ ions to maximal values of V= 235.5+/-11.8 nmol.min(-1).mg(-1) and V=234.8+/-7.0 nmol.min(-1).mg(-1), respectively. In contrast to ATP hydrolysis, K(+)-phosphatase activity was not synergistically stimulated by NH4+ and K+ ions. These data suggest that at high NH4+ ion concentrations, the (Na+, K+)-ATPase exposes a new site; the subsequent binding of NH4+ ions stimulates ATP hydrolysis to rates higher than those for K+ ions alone. This is the first demonstration that (Na+, K+)-ATPase activity in a freshwater shrimp gill is modulated by ammonium ions, independently of K+ ions, an effect that may constitute a fine-tuning mechanism of physiological relevance to osmoregulatory and excretory processes in palaemonid shrimps.