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

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

Valinomycin-induced chloride permeability in isolated rat liver mitochondria

1. Ionophore-induced osmotic swelling was used to study Cl- transport in isolated rat liver mitochondria. 2. Energy-dependent, neutral ionophore-induced swelling in Cl- salts at pH 7.2 required K+ and was preceded by a brief lag phase that was absent in chlorotributyltin-induced swelling. 3. Treatments that stimulated or inhibited mitochondrial K+/H+ exchange had qualitatively similar effects on both valinomycin-induced swelling and the associated lag phase. 4. The results suggest that valinomycin-induced Cl- permeability results from an interaction between the K+/H+ antiporter and neutral ionophore K+ complexes.     

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.     

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.     

The effects of chlorotetracycline on calcium movements in isolated rat liver mitochondria

Chlorotetracycline was used as a fluorescent chelate probe for visualizing calcium movements in rat liver mitochondria. It was demonstrated that under specified conditions, chlorotetracycline-associated fluorescence may be employed as a monitor of calcium uptake by mitochondrial membranes, e.g., at low calcium and Chlorotetracycline concentrations and in the absence of exogenous phosphate or acetate. However, at elevated calcium concentrations, e.g., >0.05 mm, a transient fluorescence response was observed upon addition of calcium to energized mitochondria. This transient or cyclic behavior of the chlorotetracycline-associated fluorescence was minimized by increasing the chlorotetracycline concentration, the mitochondrial protein concentration, or by including magnesium in the incubation. Also, it was demonstrated that chlorotetracycline addition to mitochondria which had been loaded previously with ⁴⁵Ca resulted in a rapid efflux of the accumulated ⁴⁵Ca. Because of the various effects of chlorotetracycline on the ability of the mitochondria to accumulate and to retain calcium, caution must be exercised in the interpretation of experimental results when this fluorescent chelate probe is utilized to monitor the association of divalent metal cations with biological membranes.     

The effects of cryopreservation on protein synthesis and membrane transport in isolated rat liver mitochondria.

Protein synthesizing activity and membrane transport were examined in fresh and cryopreserved isolated rat liver mitochondria. In the presence of 0.6, 1.2, and 1.8 M final concentrations of dimethyl sulfoxide (Me2SO), both metabolic parameters were considerably inhibited in the fresh samples and even more inhibited in the cryopreserved specimens. However, simple exposure to this penetrating cryoprotectant, followed by its subsequent removal by washing, did not seem to affect significantly the examined functions. When different freeze-thaw regimes were investigated, it was observed that optimal recovery of protein synthesis and membrane transport functions were obtained when fast freezing took place in the absence of Me2SO.     

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.     

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions

Summary.  The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V_max at glutamine concentration of ≤5 mM, and increased K_m for substrate concentration above 5 mM. The activation of the enzyme by bicarbonate was through an increase in V_max. Ammonium and bicarbonate ions may therefore be important physiological regulators of PDG. It is suggested that phosphate and other polyvalent ions may function by preventing product inhibition of the enzyme through promotion of PDG dimer formation. The dimerized enzyme may have a high affinity for glutamine and reduced sensitivity to inhibition by ammonium ions. Received August 10, 2001 Accepted April 1, 2002 Published online August 30, 2002 Acknowledgement This work was supported by University of Zimbabwe research grant to Dr. B. Masola. Authors' address: Dr. Bubuya Masola, Department of Biochemistry, University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe, E-mail: masolab@yahoo.co.uk     

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

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

Factors influencing the activity of ornithine aminotransferase in isolated rat liver mitochondria.

1. The characteristics of ornithine catabolism by the aminotransferase pathway in isolated mitochondria were determined. 2. Ornithine synthesis from glutamate and glutamate gamma-semialdehyde produced by the oxidation of proline was studied. No ornithine was formed in the absence of rotenone. 3. The mechanism of ornithine transport was reinvestigated, and the existence of an ornithine+/H+ exchange system postulated. 4. The kinetics of ornithine transport, ornithine catabolism in intact mitochondria and ornithine aminotransferase activity in solubilized mitochondria were compared. It is concluded that ornithine aminotransferase activity in liver mitochondria is rate-limited by the transport of ornithine across the mitochondrial membrane, and that this enzyme is involved primarily in ornithine degradation rather than ornithine synthesis.     

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.     

Allosteric properties of phosphate-activated glutaminase of human liver mitochondria

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of 2H2O. The yields were increased in association with the elevation of the 2H2O concentration. 2H2O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of 2H2O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the 2H2O concentration. The enhancement of the polymerization of tubulin by 2H2O could, therefore, be the result of the strengthening of intra- and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in length and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with 2H2O may be caused by the direct involvement of 2H2O in the polymerization of tubulin.     

The effects of tetrachlorobiphenyls on the electron transfer reaction of isolated rat liver mitochondria

A comparative study was made of the effects of several symmetrical tetrachlorobiphenyls (TCBs) on the electron transfer from succinate to oxygen of rat liver mitochondria, and some differences in effects caused by the different chlorine positions of the biphenyl ring were clarified. TCBs used in this study included 2,3,2',3'-, 2,4,2',4'-, 2,5,2',5'-, 2,6,2',6'-, and 3,4,3',4'-TCBs. The inhibitory actions of 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs on succinate oxidase were potent, while those caused by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly weak. The inhibition sites of 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs in succinate oxidase were succinate dehydrogenase and cytochrome b-c segment of the electron transport chain. In the cytochrome b-c segment, these TCBs acted on myxothiazol-sensitive site rather than antimycin-sensitive site. Cytochrome c oxidase was hardly affected by TCBs. These results indicate that 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs severely depress the electron transfer with succinate as the substrate, which secondarily reduces the synthesis of ATP. The relationship between the activity and chemical structure of TCBs is also discussed.     

Response of rat liver glutaminase to pH. Mediation by phosphate and ammonium ions

The activity of rat liver glutaminase from sedimented fractions of freeze-thawed mitochondria is strongly affected by variation in pH over a physiologically relevant range at approximate physiological concentrations of activators. As pH increases from 7.1 to 7.7 at 0.7 mM ammonium and 10 mM phosphate, the S0.5 for glutamine decreases 3.5-fold, from 38 to 11 mM. This results in an 8-fold increase in reaction velocity at 10 mM glutamine. In addition, the M0.5 for phosphate activation decreases from 21 to 8.9 mM as pH increases from 7.1 to 7.7. This apparent effect of pH on the affinity of glutaminase for phosphate is similar to previous reports of the pH effect on activation by ammonium (Verhoeven, A. J., Van Iwaarden, J. F., Joseph, S. K., and Meijer, A. J. (1983) Eur. J. Biochem. 133, 241-244; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 159, 296-302). Glutaminase does not respond to variation in pH between 7.1 and 7.7 when phosphate and ammonium are saturating. The effects of the two modifiers are additive. Each is still effective, as is pH, when the other is saturating. Therefore, it appears that the effects of pH on the apparent affinity of the enzyme for ammonium and phosphate account for the enzyme's response to pH. These results may help explain previous reports of minimal effects of pH on glutaminase at saturating concentrations of related substances (McGivan, J. D., Lacey, J. H., and Joseph, K. (1980) Biochim. J. 192, 537-542; Horowitz, M. L., and Knox, W. E. (1968) Enzymol. Biol. Clin. 9, 241-255; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 759, 296-302). Glutaminase binds glutamine cooperatively with Hill coefficients ranging from 1.7 to 2.2, which suggests at least two and probably three or more interacting binding sites for glutamine. The strong response of liver glutaminase to pH and the fact that the reaction can supply metabolites for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

Immunoblot analysis of glutaminase peptides in intact and solubilized mitochondria isolated from various rat tissues.

Antibodies were prepared against isolated rat renal glutaminase and affinity-purified against the 65 kDa peptide contained in the purified rat brain glutaminase. The affinity-purified IgGs were then used to compare the glutaminase immunoreactive peptides contained in samples that had been subjected to SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose. The purified brain glutaminase and isolated brain mitochondria contain 68 and 65 kDa peptides that exhibit nearly equivalent immunostaining. Partial proteolysis of the isolated 68 and 65 kDa peptides with Staphylococcus aureus V8 proteinase produced an identical pattern of immunoreactive proteolytic fragments. However, digestion of the two peptides with chymotrypsin resulted in similar, but slightly different, patterns. The pattern of immunostaining was unaltered even when the brain mitochondria were solubilized with Triton X-100 and stored for 2 days at 4 degrees C. A very similar pattern was observed when intact renal mitochondria were subjected to immunoblot analysis. However, when renal mitochondria were solubilized, the 68 kDa peptide was rapidly degraded to the 65 kDa form. At 4 degrees C this reaction occurs with apparent first-order kinetics and a t1/2 of 35 min. Degradation of the 65 kDa form of the renal glutaminase occurs with much slower kinetics, but is nearly complete after 24 h. Solubilization of mitochondria isolated from various zones of the kidney indicated that the responsible endogenous proteinase was localized primarily in the cortex. Mitochondria isolated from intestinal or renal papillary tissue contain four glutaminase immunoreactive peptides (Mr 68,000, 65,000, 61,000 and 58,000). The smallest of these peptides is identical in size with the single immunoreactive peptide observed in liver tissue.