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.     

Effects of mitochondrial swelling and calcium on phosphate-activated glutaminase in pig renal mitochondria

The effects of mitochondrial swelling and calcium have been used to study the possible function of the glutamine transporter in regulating glutamine hydrolysis. Salt-induced swelling of pig renal mitochondria and an iso-osmotic mixed salt solution and swelling caused by reducing the osmolarity of the incubation medium, are accompanied by activation of glutamine hydrolysis. Regulation of the glutaminase activity by salt-induced mitochondrial swelling is likely to have physiological importance, similar to the regulation of hepatic glutaminase by changing the matrix volume, that has been described by others. 0.1-1.0 mM calcium stimulates glutamine hydrolysis and the calcium activation curve follows Michaelis-Menten kinetics. The calcium activation is reversible, it is unaffected by phosphate, high glutamine and mitochondrial calcium uptake, as well as by sonication and the activation is calmodulin independent. The calcium activation is additive to that of swelling. Similar to calcium, hypo-osmotic swelling mainly increases the apparent Vmax for glutamine, whereas the apparent Km is little changed, indicating that the effects are primarily on the phosphate-activated glutaminase itself rather than on the glutamine transporter. Furthermore, calcium which activates glutamine hydrolysis, inhibits glutamine uptake into the mitochondria and so does alanine having no effect on glutamine hydrolysis. Therefore, it is indicative that glutamine transport is not rate limiting for glutamine hydrolysis.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine

The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated.

1. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates.

2. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane.

3. (3) Mersalyl (plus CCCP) markedly decreased [¹⁴C]glutamine- and [³²P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found.

4. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme.

On the basis of these findings it is concluded that there is an association between the transport of phosphate on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existence of a specific glutamine-phosphate carrier is not ruled out.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase.

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH4+-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibted by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitos of respiration or an uncoupler but not in ther absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition. On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibted by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Response of rat liver glutaminase to pH, ammonium, and citrate. Possible regulatory role of glutaminase in ureagenesis

Liver glutaminase is stimulated by an increase in NH4+ concentration and NH4+ is an absolute requirement for activity at approximate physiological concentrations of phosphate and glutamine. Increases in the concentration of NH4+ cannot, however, overcome the inhibitory effect of a decrease in pH. In addition, the concentration of NH4+ required for half-maximal rate decreases as pH increases. This decrease is the result of two factors: a direct effect of pH on the apparent affinity of the enzyme for NH4+, and an indirect effect of pH brought about by an increase in the apparent affinity of the enzyme for phosphate which results in a further decrease in the M0.5 for NH4+. In addition, liver glutaminase responds strongly to the concentration of citrate over a physiologically relevant range at approximate physiological concentrations of NH4+, phosphate, and glutamine. An increase in citrate concentration stimulates glutaminase by increasing the affinity of the enzyme for glutamine. The apparent affinity of the enzyme for citrate increases as pH increases. The strong response of liver glutaminase to pH, NH4+, and citrate and the fact that the hydrolysis of glutamine can supply metabolites and effectors for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

The effects of ammonium chloride and bicarbonate on the activity of glutaminase in isolated liver mitochondria.

1. Glutamine hydrolysis in liver mitochondria was studied by measuring the production of glutamate under conditions where this compound could not be further metabolized. 2. Glutaminase activity in intact mitochondria was very low in the absence of activators. 3. Glutamine hydrolysis was markedly stimulated by NH4Cl and also by HCO3- ions. 4. The stimulation by each of these compounds was much decreased if the mitochondria were uncoupled. 5. Maximum rates of glutamine hydrolysis required the addition of phosphate. A correlation was observed between the activity of glutaminase in the presence of NH4Cl plus HCO3- and the intramitochondrial content of ATP. 6. In disrupted mitochondria, NH4Cl stimulated glutaminase to a much smaller extent than in intact mitochondria. The NH4Cl stimulation in disrupted mitochondria was much increased by the addition of ATP. KHCO3 also stimulated glutaminase activity in disrupted mitochondria, and ATP increased the magnitude of this stimulation. 7. It was concluded that maximum rates of glutaminase activity in liver mitochondria require the presence of phosphate, ATP and either HCO3- or NH4+. A comparison of the results obtained on intact and broken mitochondria indicates that these effectors have a direct effect on the glutaminase enzyme system rather than an indirect effect mediated by changes in transmembrane ion gradients or in the concentrations of intramitochondrial metabolites.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH₄⁺-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate.

1. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue.

2. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibited by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate.

3. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitors of respiration or an uncoupler but not in their absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition.

On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibited by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Conditions for activity of glutaminase in kidney mitochondria

1. Rat kidney mitochondria oxidize glutamate very slowly. Addition of glutamine stimulates this respiration two- to three-fold. Addition of glutamate also stimulates respiration in the presence of glutamine. 2. By measuring mitochondrial swelling in iso-osmotic solutions of glutamine or of ammonium glutamate it was shown that glutamine penetrates the mitochondrial membrane rapidly whereas ammonium glutamate penetrates very slowly. 3. Experiments in which reduction of NAD(P)⁺ was measured in preparations of intact and broken mitochondria indicated that glutamate dehydrogenase shows the phenomenon of `latency'. On the addition of glutamine rapid reduction of nicotinamide nucleotides in intact mitochondria was obtained. 4. During the action of glutaminase there is an accumulation of glutamate inside the mitochondria. 5. When the mitochondria were suspended in a medium containing glutamine, P_i and rotenone the rate of production of ammonia was stimulated by the addition of a substrate, e.g. succinate. Addition of an uncoupler or antimycin A abolished this stimulation. 6. The effects of succinate and uncoupler were especially pronounced in the presence of glutamate, which is an inhibitor of glutaminase activity by competition with P_i. 7. Determination of the enzyme activity in media at different pH values showed that the optimum pH for glutaminase activity in the preparation of broken mitochondria was 8, whereas for intact mitochondria it was dependent on the energy state. In the presence of succinate as an energy source it was pH 8.5, but in the presence of uncoupler or antimycin A it was 9. This displacement of the pH optimum to a higher value was especially pronounced in the presence of both glutamate and uncoupler. 8. If nigericin was present in potassium chloride medium the pH optimum for enzyme activity in intact non-respiring mitochondria was nearly the same as in the preparation of broken mitochondria; however, its presence in K⁺-free medium displaced the pH optimum for glutaminase activity to a very high value. 9. It is postulated that because of low permeability of the kidney mitochondrial membrane to glutamate the latter accumulates inside the mitochondria, and that this leads to the inhibition of the enzyme by competition with P_i and also by lowering the pH of the intramitochondrial space. With succinate as substrate for respiration there is an outward translocation of H⁺ ions, which together with accumulation of P_i increases glutaminase activity. Translocation of K⁺ ions inward increases the enzyme activity, perhaps by increasing the pH of the internal spaces and causing an accumulation of P_i. 10. The importance of the location of the enzyme in the mitochondria in relation to its biological function and conditions for activity is discussed.     

Location of an oligomycin-insensitive and magnesium ion-stimulated adenosine triphosphatase in rat spleen mitochondria.

1. When rat spleen mitochondria are incubated with oxidizable substrates, added MgCl2 (greater than 150 muM free concentration) markedly stimulates state-4 respiration and lowers both the respiratory control and ADP/O ratios; this effect is reversible on addition of excess of EDTA. 2. With [gamma-32P]ATP as substrate, an Mg2+-stimulated ATPase (adenosine triphosphate) was identified in the atractyloside-insensitive and EDTA-accessible space of intact rat spleen mitochondria. 3. Oligomycin has no effect on the activity of the Mg2+-stimulated ATPase at a concentration (2.0mug/mg of protein) that completely inhibits the atractyloside-sensitive reaction. Of the two ATPase activities, only the atracytoloside sensitive reaction is stimulated (approx. 40%) by dinitrophenol. 4. On digitonin fractionation the atractyloside-insensitive Mg2+-stimulated ATPase co-purifies with the outer membrane-fraction of rat spleen mitochondria, whereas (as expected) the atractylosidesensitive activity co-purifies with the inner-membrane plus matrix fraction. 5. Stoicheiometric amounts of ADP and Pi are produced as the end products of ATP hydrolysis by purified outer-membrane fragments; no significant AMP production is detected during the time-course of the reaction. 6. The outer-membrane ATPase is present in rat kidney cortex and heart mitochondria as well as in spleen, but is absent from rat liver, thymus, brain, lung, diaphragm and skeletal muscle.     

K(+)- and Mg2(+)-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg2+ and EGTA and is stimulated by Ca2+. The Mg2(+)-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5 mM MgCl2, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca2+ causes no further increase in the rate of hydrolysis and Ca2+ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca2+ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by Pi unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect Pi inhibition of the Mg2(+)-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a Pi-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and Pi. In this conformation the enzyme is transformed from a Ca2(+)- and Mg2(+)-dependent ATPase into a (K+ + Mg2+)-ATPase.     

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.     

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.     

Effect of different dietary proteins on plasma and liver fatty acid compositions in growing rats

The activities of key glutamine and urea cycle enzymes were assayed in liver homogenates from control and chronically acidotic rats and compared with citrulline and urea productions by isolated mitochondria and intact liver slices, respectively. Glutamine-dependent urea and citrulline synthesis were increased significantly in isolated mitochondria and in liver slices; the activities of carbamoyl phosphate synthetase and arginase were unchanged and increased, respectively. Glutamine was not a precursor in the carbamoyl phosphate synthetase system, suggesting that the glutamine effect is an indirect one and that glutamine requires prior hydrolysis. Increased mitochondrial citrulline synthesis was associated with enhanced oxygen consumption, suggesting glutamine acts both as a nitrogen and fuel source. Hepatic phosphate-dependent glutaminase was elevated by chronic acidosis. The results indicate that the acidosis-induced reduction in ureagenesis and reversal from glutamine uptake to release observed in vivo are not reflections of corresponding changes in the hepatic enzyme content. Rather, when available, glutamine readily supports ureagenesis, suggesting a close coupling of hepatic glutaminase flux with citrulline synthesis.     

Rat liver glutaminase. Regulation by reversible interaction with the mitochondrial membrane

Partially purified rat liver mitochondrial glutaminase shows a sigmoidal dependence on glutamine concentration, and an absolute requirement for inorganic phosphate as activator. Reconstitution with a mitochondrial membrane fraction changes the kinetic properties of the enzyme making the glutamine dependence more hyperbolic and reducing the concentration of phosphate required for half-maximum activation. Glutaminase activity in isolated mitochondria is known to be increased as a result of mitochondrial swelling. In mitochondria suspended in isotonic medium, the properties of glutaminase resemble of the isolated enzyme while in swollen mitochondria the kinetic properties revert to those exhibited by the enzyme in association with the mitochondrial membrane. It is postulated that mitochondrial glutaminase is regulated in situ by reversible association with the inner mitochondrial membrane which is mediated by mitochondrial swelling. This mechanism may explain the short-term hormonally induced activation of the enzyme observed in isolated hepatocytes.     

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.     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Calcium and magnesium ions as effectors of adipose-tissue pyruvate dehydrogenase phosphate phosphatase

The metal-ion requirement of extracted and partially purified pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat-pads was investigated with pig heart pyruvate dehydrogenase [(32)P]phosphate as substrate. The enzyme required Mg(2+) (K(m) 0.5mm) and was activated additionally by Ca(2+) (K(m) 1mum) or Sr(2+) and inhibited by Ni(2+). Isolated fat-cell mitochondria, like liver mitochondria, possess a respiration- or ATP-linked Ca(2+)-uptake system which is inhibited by Ruthenium Red, by uncouplers when linked to respiration, and by oligomycin when linked to ATP. Depletion of fat-cell mitochondria of 75% of their total magnesium content and of 94% of their total calcium content by incubation with the bivalent-metal ionophore A23187 leads to complete loss of pyruvate dehydrogenase phosphate phosphatase activity. Restoration of full activity required addition of both MgCl(2) and CaCl(2). SrCl(2) could replace CaCl(2) (but not MgCl(2)) and NiCl(2) was inhibitory. The metal-ion requirement of the phosphatase within mitochondria was thus equivalent to that of the extracted enzyme. Insulin activation of pyruvate dehydrogenase in rat epididymal fat-pads was not accompanied by any measurable increase in the activity of the phosphatase in extracts of the tissue when either endogenous substrate or (32)P-labelled pig heart substrate was used for assay. The activation of pyruvate dehydrogenase in fat-pads by insulin was inhibited by Ruthenium Red (which may inhibit cell and mitochondrial uptake of Ca(2+)) and by MnCl(2) and NiCl(2) (which may inhibit cell uptake of Ca(2+)). It is concluded that Mg(2+) and Ca(2+) are cofactors for pyruvate dehydrogenase phosphate phosphatase and that an increased mitochondrial uptake of Ca(2+) might contribute to the activation of pyruvate dehydrogenase by insulin.     

Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta. Effect of substrates and potassium

This study evaluates the effect of Mg2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K+, and different oxidizable substrates. Mg2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K+, succinate and 1mM Mg2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM     

The effect of divalent cations on aggregation of Dictyostelium discoideum.

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20--40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 X 10(-5) M progesterone considerably delays it. These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid. Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+. These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.