Glutamate dehydrogenase activation and ammonia formation by rat kidney mitochondria.

Abstract

Mitochondria from rat renal cortex were incubated with physiological concentrations of glutamate, or glutamine, or both. Changes in medium ammonia, glutamate, glutamine, and aspartate were measured and fluxes through glutamate dehydrogenase, glutaminase, and glutamate oxalacetate transaminase were calculated at different pH levels in vitro and in mitochondria from animals in which metabolic acidosis was induced in vivo. In the presence of glutamate alone as substrate, aspartate formation was always large with respect to ammonia formation. Lowering the pH from 7.4 to 6.8 decreased aspartate production but resulted in no consistent increase in ammonia formation. At pH 7.4 the deamination pathway accounted for 21% and, at pH 6.8, 26% of the total glutamate utilization. When glutamine was the only substrate and glutamate was generated inside the mitochondria, the deamination pathway accounted for 39% of the glutamate utilization at pH 7.4 and 32% at pH 6.8. At pH 7.4 fluxes through glutaminase, glutamate dehydrogenase, and glutamate oxalacetate transaminase were all high with respect to the fluxes measured at pH 6.8. With both glutamine and glutamate as substrates, glutamate metabolism occurred almost exclusively by transamination and the deamination pathway was negligible. Ammonia generation by mitochondria isolated from animals subjected to 7 days of NH&l acidosis was increased 235% in the presence of both substrates. Glutamate deamination was markedly accelerated and contributed one-half of the augmented ammonia synthesis. With glutamine as substrate, glutamate dehydrogenase flux increased 4-fold while glutaminase flux rose a-fold. In the presence of both glutamine and glutamate, the increase in glutamate dehydrogenase flux was even more marked, rising 50-fold compared to control. The increase in glutamate dehydrogenase flux could not be explained entirely by an increase in glutamate dehydrogenase enzyme activity, enhanced glutamate transport, an alteration in the mitochondrial oxidation-reduction state or by equilibrium considera