Demonstration of a Requirement of High Energy Phosphate for the Aerobic Oxidation of Succinate in Liver Mitochondria

It was concluded in a previous paper (1) that the enhancement of respiration by inorganic orthophosphate, occurring in rat liver mitochondria oxidizing a number of Krebs cycle metabolites or glutamate in the presence of 2,4-dinitrophenol or Dicumarol, is due to the substrate level phosphorylation linked to the oxidation of a-ketoglutarate which is not uncoupled by these agents. Yet, no requirement of inorganic orthophosphate under these conditions was observed when succinate was used as a substrate. It was observed in the course of these studies, however, that when the mitochondria were pretreated’ with dinitrophenol plus adenosine 5’-phosphate, in order to remove endogenous inorganic orthophosphate, and the initial rate of respiration after incubation with succinate and dinitrophenol was measured with the oxygen electrode, this was markedly low, unless inorganic orthophosphate was included in the incubating medium. Moreover, it was found necessary to add the inorganic orthophosphate together with the succinate some time before the addition of dinitrophenol, in order to obtain a high rate of respiration. Alternatively, succinate oxidation could be brought to a high rate by adding adenosine triphosphate to the system, and in this case even after the addition of dinitrophenol. Similar effects were subsequently obtained, and even in a more striking manner, when the mitochondria were preincubated with arsenate, instead of the pretreatment with dinitrophenol plus adenosine 5’-phosphate. Studies with PVabeled mitochondrial preparations have revealed that treatment of mitochondria both with dinitrophenol plus adenosine 5’-phosphate (1) and with arsenate (2) results in an extensive depletion of not only endogenous inorganic orthophosphate but also endogenous high energy phosphate. The above findings were therefore interpreted to suggest that the mitochondrial oxidation of succinate might require a supply of high energy phosphate. Experimental evidence in favor of this concept has been reported briefly (3), and is presented here in detail.