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Mitochondria from dog renal cortex were incubated with L-[14Cglutamine. Glutamate metabolism was prevented by inhibitors so that glutamate accumulated either in the mitochondrial matrix space or in the medium. The formation and accumuation of glutamate formed from glutamine and the distribution of glutamine in the mitochondrial fluid spaces were studied. In the matrix space glutamate rapidly reaches levels over 5 times that of glutamine in the medium. A more gradual accumulation occurs in the medium as glutamate is transported out of the mitochondria. Addition of an energy source such as succinate to the medium accelerates glutamate formation. A Km of 0.6 mM appears to govern the reaction at low concentrations of glutamine; at about 4 mM an abrupt change kinetics occurs with a Km of 5 mM above that level. Both NH4+ and glutamate inhibit glutamine metabolism and phosphate stimulates it, but little …