Ammonia Formation in Isolated Rat Liver Mitochondria*

Studies of isolated rat liver mitochondria were undertaken in order to evaluate the importance of glutama te transport, oxidation reduction state, and product inhibition on the rates of formation of ammonia from glutamate. Uptake and efflux of glutamate across the mitochondrial membrane were measured isotopically in the presence of rotenone. Efflux was stimulated by H+ in the mitochondrial matrix and was found to be first order with respect to matrix g lu tamate except when the mat r ix pH was unphysiologically low. The data suggest that the K,,, of matrix glutamate for efflux is decreased b y H+. Matrix H+ also appeared to stimulate glutamate uptake, but the effect was to increase both the K,,, of medium glutamates and Vmm. Mitochondria were incubated at 15 and 28 “C with glutamate and malonate. Under these conditions, glut ama te was metabolized only b y the deamination pathway. Flux was evaluated by assay of ammonia formation. Oxidation reduction state was varied with ADP and uncoupling agents. Matrix a-ketoglutarate was varied either b y the omission of malonate from the incubation media or by adding a-ketoglutarate to the external media. Influx and efflux of glutamate could be calculated from previously determined transport parameters. The difference between calculated influx and efflux was found to be equal to ammonia formation under all conditions. It was, therefore, possible to evalua te the relative contributions of oxidation reduction state, transport, and product inhibition as effectors of ammonia formation. The contribution of transport was relatively small while oxidation reduction state exerted a large influence. a-Ketoglutarate was found to be a potent competitive inhibitor of ammonia product ion and glutamate dehydrogenase. Inhibition of g lu tamate dehydrogenase by a-ketoglutarate was judged to be a potentially important modulator of metabolic fluxes.