NAD(P)-LINKED OXIDOREDUCTIONS AND NICOTINAMIDE NUCLEOTIDE SPECIFICITY OF GLUTAMATE DEHYDROGENASE IN RAT-LIVER MITOCHONDRIA

1. 1. The oxidoreduction pattern of intramitochondrial NAD(P) during the transfer of hydrogens from α-oxoglutarate to α-oxoglutarate (+ ammonia), oxaloacetate, acetoacetate, and α-oxoglutarate (+ CO2) in rat-liver mitochondria was studied. The intramitochondrial NAD(P) was first subjected to a cycle of oxidation by O2 and reduction by α-oxoglutarate, either in the coupled state (ADP + Pi present) or in the uncoupled state (dicoumarol + oligomycin present), before adding the hydrogen acceptor. 2. 2. When the cycle of oxidation and reduction was carried out in the coupled state, NADP was reduced to a greater extent than NAD. When it was done in the uncoupled state, NAD was more reduced than NADP. 3. 3. In the coupled mitochondria, the oxidation rate of NADPH by α-oxoglutarate (+ ammonia) at 10° was higher than that of NADH. In the uncoupled mitochondria, the rate of oxidation of NADPH was greatly inhibited, and that of NADH only slightly. 4. 4. With acetoacetate as hydrogen acceptor, NADH was oxidized rapidly and NADPH only slowly (at 10°), both in the coupled and in the uncoupled state. Similar results were obtained with oxaloacetate. 5. 5. When the hydrogen acceptor was α-oxoglutarate (+ CO2), both NADH and NADPH were rapidly oxidized (at 25°) in the coupled mitochondria. In uncoupled mitochondria, no oxidation of NADH occurred initially upon the addition of α-oxoglutarate (+ CO2), and furthermore, the oxidation of NADPH was inhibited. 6. 6. The effect of carrying out the oxidation part of the cycle in the uncoupled state was shown to be reversible. When dicoumarol was removed with albumin, and the addition of α-oxoglutarate could provide not only reducing equivalents, but also energy, the mitochondria behaved exactly like those preincubated in the coupled state. 7. 7. It is concluded that when hydrogen is transferred from an NAD-linked substrate to α-oxoglutarate (+ ammonia or CO2), energy is required not only to promote the transhydrogenation between NADH and NADP+, but also for the reaction of NADPH with glutamate dehydrogenase or the NADP-linked isocitrate dehydrogenase. Energy is not required for the oxidation of NADH by acetoacetate or oxaloacetate. A possible mechanism is proposed. 8. 8. The physiological significance of the results is discussed. © 1969.