Control of nicotinamide nucleotide-linked oxidoreductions in rat-liver mitochondria

Abstract

1. 1. With α-oxoglutarate as the hydrogen donor and the substrate-linked phosphorylation step as the source of energy, hydrogen transfer to α-oxoglutarate ( plus ammonia) in rat-liver mitochondria was inhibited by dicoumarol or oligomycin, and concomitantly, intramitochondrial NAD became more reduced. Hydrogen transfer to α-oxoglutarate ( plus CO 2) was also inhibited by the uncoupler, but not that to acetoacetate or oxaloacetate. 2. 2. Hydrogen transfer from β-hydroxybutyrate to α-oxoglutarate ( plus ammonia) was stimulated by ATP. The stimulation was oligomycin-sensitive. 3. 3. Hydrogen transfer from glutamate to acetoacetate or oxaloacetate was inhibited by ATP. This inhibition was oligomycin-sensitive. 4. 4. With isocitrate as hydrogen donor, the reduction of acetoacetate was slightly stimulated by ATP. Reduction to α-oxoglutarate ( plus ammonia) was not affected by ATP or by dicoumarol. 5. 5. The reduction of acetoacetate or oxaloacetate with glutamate as hydrogen donor was inhibited by ATP, and concomitantly, NADP became more reduced. The effect of ATP was oligomycin-sensitive. 6. 6. The effect of the mitochondrial energy level on the reductive amination of α-oxoglutarate in rat-liver mitochondria with α-oxoglutarate as the hydrogen donor was studied in detail. Following a preincubation in order largely to oxidize the intramitochondrial nicotinamide nucleotides, the overall reaction was separated into two steps: the reduction of intramitochondrial NAD(P) + by α-oxoglutarate, and the oxidation of the NAD(P)H formed by α-oxoglutarate ( plus ammonia). 7. 7. When dicoumarol plus oligomycin was present during the preincubation, the subsequent oxidation of intramitochondrial NAD(P)H by α-oxoglutarate ( plus ammonia) was strongly inhibited. The inhibition could be prevented by removal of the dicoumarol after the preincubation and allowing α-oxoglutarate to be oxidized via the respiratory chain. 8. 8. Preincubation with dicoumarol plus oligomycin led to inhibition of the accumulation of α-oxoglutarate by the mitochondria. However, a kinetic analysis showed that diminished substrate penetration was not the cause of the inhibition in the uncoupled mitochondria of the oxidation of intramitochondrial NAD(P)H by α-oxoglutarate ( plus ammonia). 9. 9. The results are discussed in relation to the NADP specificity of glutamate dehydrogenase in intact rat-liver mitochondria, the role of the energy-linked transhydrogenase, and a possible energy requirement for the oxidation of intramitochondrial nicotinamide nucleotides (previously reduced by NAD-linked substrates) by α-oxoglutarate ( plus ammonia).