The nicotinamide nucleotide specificity of glutamate dehydrogenase in intact RAT-liver mitochondria

1. The kinetics of oxidation of intramitochondrial reduced nicotinamide nucleotides by -oxoglutarate plus ammonia in intact rat-liver mitochondria have been reinvestigated. It is demonstrated that the preferential oxidation of NADPH observed on addition of ammonia to mitochondria, preincubated under energized conditions in the presence of -oxoglutarate, is due to a transhydrogenation catalysed by glutamate dehydrogenase rather than to an energy-dependent modification of the nicotinamide nucleotide specificity of the enzyme in intact mitochondria.

2. When mitochondria are preincubated at 25 °C under energized conditions in the presence of respiratory inhibitors with the substrates of glutamate dehydrogenase, an oxidation of NADPH, but not of NADH, is brought about by decreasing the reaction temperature. Both the rate of NADPH oxidation and the final steady-state mass-action ratio of nicotinamide nucleotides are dependent on the concentration of ammonia and on the final reaction temperature. A similar effect is observed when rhein is added to the reaction medium at 25 °C in order to inhibit the energy-linked transhydrogenase reaction.

3. In the presence of the substrates of glutamate dehydrogenase, intact ratliver mitochondria catalyse an ATPase reaction due to the simultaneous activity of the energy-linked transhydrogenase and the non-energy-linked transhydrogenation catalysed by glutamate dehydrogenase.

4. These findings are discussed in relation to the nicotinamide nucleotide specificity of glutamate dehydrogenase and to a possible compartmentation of nicotinamide nucleotides in intact rat-liver mitochondria.