Kinetics of Phosphorylation of Intramitochondrial and Extramitochondrial Adenine Nucleotides As Related to Nucleotide Translocation

Abstract

The kinetics of oxidative phosphorylation of extramitochondrial (external) ADP and intramitochondrial (internal) ADP and AMP has been studied under conditions required for the exchange-diffusion of adenine nucleotides through mitochondrial membranes (ethylenediaminetetraacetate-supplemented medium at 0°) and under conditions favoring the leakage of internal adenine nucleotides (Mg++-supplemented medium). By means of atractyloside, it is possible to show that under conditions of exchange-diffusion the kinetics of phosphorylation of internal ADP and AMP is not altered by the addition of ADP to mitochondria and is independent of the phosphorylation of external ADP. Internal ADP and AMP are converted into ATP through a three-step process which successively involves first a rapid oxidative phosphorylation of the internal ADP and ATP, second a transient plateau phase, and finally a conversion of AMP into ATP. The oxidative phosphorylation of external ADP starts after a lag period which corresponds to the time interval required for the oxidative phosphorylation of the internal ADP into ATP. The conversion of the internal AMP into ATP proceeds by two coupled reactions, namely an ATP-dependent transphosphorylation of AMP into ADP and the oxidative phosphorylation of ADP into ATP. As suggested by the data of 32P-labeling experiments, the enzymatic systems catalyzing the two above mentioned coupled reactions are structurally and functionally linked to form a complex which catalyzes the over-all conversion of AMP into ATP in a cyclic manner. The ATP which is the phosphate donor for the transphosphorylation reaction is not the free ATP pre-existing in mitochondria, but the ATP produced by the complex itself; on the other hand, the ADP formed as a transient intermediate in the AMP-ATP conversion is immediately phosphorylated to give ATP without mixing with the free internal ADP. These data point to a compartmentalization of the enzymatic complex involved in the AMP-ATP conversion. Addition of magnesium ions to mitochondria incubated in a standard medium for oxidative phosphorylation (supplemented with O2, phosphate, and oxidizable substrate) results in a leakage of internal adenine nucleotides. This leakage is slowed by inhibitors or uncouplers of oxidative phosphorylation or by a low temperature, but not by atractyloside; ATP is preferentially released compared to ADP and AMP. The adenine nucleotides released outside mitochondria are rapidly exchanged with those remaining inside mitochondria. Since the exchange is much faster than the release, the distribution patterns of the adenine nucleotides remaining inside mitochondria or released outside mitochondria are similar; for instance under the conditions of oxidative phosphorylation, the same high level of ATP is found inside and outside mitochondria. The effect of atractyloside on the apparent efficiency of the oxidative phosphorylation of internal adenine nucleotides is discussed with respect to the above data.