Comparative studies on glutamate metabolism in synpatic and non-synaptic rat brain mitochondria

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Abstract
The apparent Michaelis constants of the glutamate dehydrogenase (EC 1.4.1.3), the glutamate-oxaloacetate transaminase (EC 2.6.1.1) and the glutaminase (EC 3.5.1.2) of rat brain mitochondria derived from non-synaptic (M) and synaptic (SM2) sources were studied. The kinetics of O2 uptake by both populations of mitochondria in the presence of a fixed concentration of malate and various concentrations of glutamate or glutamine were investigated. In both mitochondrial populations, glutamate-supported respiration in the presence of 2.5 mM malate appeared to be biphasic, 1 system (B) having an apparent Km for glutamate of 0.25 .+-. 0.04 mM (n = 7) and the other (A) of 1.64 .+-. 0.5 mM (n = 7) [when corrected for low-Km process, Km = 2.4 .+-. 0.75 mM (n = 7)]. Aspartate production in these experiments followed kinetics of a single process with an apparent Km for glutamate of 1.8-2mM, approximating to the high Km process. O2-uptake measurement with both mitochondrial populations in the presence of malate and various glutamate concentrations in which amino-oxyacetate was present showed kinetics approximating only to the low-Km process (apparent Km for glutamate .apprx. 0.2 mM). Similar experiments in the presence of glutamate alone showed kinetics approximating only to the high-Km process (apparent Km for glutamate .apprx. 1-1.3mM). O2 uptake supported by glutamine (0-3 mM) and malate (2.5 mM) by the free (M) mitochondrial population showed single-phase kinetics with an apparent Km for glutamine of 0.28 mM. Asparate and 2-oxoglutarate accumulation was measured in free non-synaptic (M) brain mitochondria oxidizing various concentrations of glutamate at a fixed malate concentration. Over a 30-fold increase in glutamate concentration, the flux through the glutamate-oxaloacetate transminase increased 7-8-fold, whereas the flux through 2-oxoglutarate dehydrogenase increased about 2.5-fold. The biphasic kinetics of glutamate-supported respiration by brain mitochondria in the presence of malate were interpreted as reflecting this change in the relative fluxes through transmination and 2-oxoglutarate metabolism.