Regulation of glutaminase by exogenous glutamate, ammonia and 2-oxoglutarate in synaptosomal enriched preparation from rat brain.

Phosphate activated glutaminase in synaptosomal enriched preparation from rat brain is very sensitive to inhibition by low concentration of glutamate, ammonia and 2-oxoglutarate when added to the incubation medium at pH 7.6. By increasing the concentration of either of these compounds up to 0.5 mM a pronounced initial inhibition is followed by little or no further effect when the concentration is increased beyond this level. By lowering the pH of the reaction mixture to 7.0, the inhibition by glutamate is almost abolished and that of ammonia reduced. Glutamate inhibits mainly the N-ethylmaleimide-sensitive fraction of glutaminase which previously is suggested to be localized to the outer phase of the mitochondrial inner membrane whereas ammonia inhibits both the N-ethylmaleimide-sensitive and -insensitive fraction. Evidence has been produced to show that the inhibition by 2-oxoglutarate is caused by glutamate formation by aminotransferase reactions. Since 2-oxoglutarate is produced by the tricarboxylic acid cycle, the operation of this cycle may regulate the glutaminase reaction by controlling glutamate formation via the aminotransferase reactions.

Evidence for compartmentalization of glutamate in rat brain synaptosomes using the glutamate sensitivity of phosphate-activated glutaminase as a functional test.

The glutamate content of rat brain synaptosomes was measured by high performance liquid chromatography to be 39 micromol/g protein. If uncompartmentalized this glutamate (4 mM) would inhibit phosphate-activated glutaminase considerably. Since the action of any endogenous effector on the enzyme is assumed to be negligible following disruption of the synaptosomes, due to dilution with the incubation medium, the inhibition by glumate and activation by phosphate were compared in intact and disrupted synaptosomes. The inhibition by endogenous glutamate in intact synaptosomes was found to correspond to less than that of 0.5 mM of added glutamate to disrupted synaptosomes, indicating that the major fraction of synaptosomal glutamate is compartmentalized.