Differences between the Reactivity of Endogenous and Exogenous Adenine Nucleotides in Mitochondria as Studied at Low Temperature

The rates of phosphorylation of endogenous and exogenous ADP and of the uncoupler stimulated hydrolysis of endogenous and exogenous ATP by rat liver mitochondria have been measured at temperatures between 2–14°. 1) The rate of the phosphorylation of endogenous ADP was found to be more than three times higher than the rate of the phosphorylation of exogenous ADP at 6°. The rates tend to approach each other at higher temperatures. 2) The rate of the carbonylcyanide phenylhydrazone stimulated dephosphorylation of endogenous ATP was more than twenty times higher than that of exogenous ATP below 6°. Even at room temperature there remains a considerable difference between the two rates. Hence, it is concluded that only the hydrolysis of endogenous ATP can be taken as a measure of the activity of mitochondrial ATPase. At low temperatures ATPase was not stimulated by dinitrophenol, and dinitrophenol caused an inhibition of carbonylcyanide phenylhydrazone stimulated ATPase. 3) Atractyloside inhibited only the reactivity of exogenous ADP and ATP, but had no influence on the rate of phosphorylation of endogenous ADP or the rate of carbonylcyanide phenylhydrazone stimulated hydrolysis of endogenous ATP. 4) In principle the same results were obtained with rat heart mitochondria, where the difference in the rate of reactivity of endogenous and exogenous adenine nucleotides was considerably smaller than in rat liver mitochondria. It is concluded that the access of exogenous adenine nucleotides to the inner compartment of the mitochondria, where the endogenous adenine nucleotides are located and where oxidative phosphorylation takes place, is limited at low temperature by the adenine nucleotide translocation.