Some Characteristics of Phosphorylation Reactions in Rat-liver Mitochondria Incubated without the Addition of Adenine Nucleotides and Their Relation to Substrate-level Phosphorylation

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Abstract
The results of 32P1-labelling of the organic phosphate fraction in rat-liver mitochondria incubated in ATP-(or ADP-) free media have been compared with those of 32P1labelling in media with ATP (or ADP) added. Differences have been noted in the sensitivity to uncouplers and inhibitors of oxidative phosphorylation, in the pH-activity profile, in the Arrhenius plot and in the 32P distribution among nucleotides. In the case of phosphorylation in the ATP-(or ADP-) free medium, significant generation of 32P-ADP is further enhanced by uncouplers, counterbalancing the uncoupler-induced inhibition of 32P-ATP formation and thus causing the 32P-labelling of the whole organic phosphate fraction to be apparently insensitive to the uncoupler. This forms a sharp contrast with the phosphorylation pattern observed in media with ATP (or ADP) added, in which there is an almost exclusive labelling of the ATP fraction. Electron acceptors, like uncouplers, increase 32P1 incorporation into ADP. The oxidation of glutamate as well as the regeneration of NAD+ from NADH is also accelerated by uncouplers and electron acceptors under exactly the same conditions. Together with the previous conclusion that the incorporation of 32P1 into ADP inside the inner membrane is closely connected with 2-oxoglutarate-linked substrate-level phosphorylation, these findings indicate that the regeneration of NAD+ caused by the transfer of electrons across the respiratory chain plays a significant role in controlling the citric acid cycle as well as coupled substrate-level phosphorylation.

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