The Interaction Between Adenine Nucleotide Transport and Phosghorylation in Intact Rat Liver Mitochondria

(0 stands for outside, in stands for inside). The specific enzyme systems are the adenine nucleotide translocator and the mitochondria1 ATPase (adenosine triphosphatase), F1. According to the experiments of Klingenberg and coworkers (Klingenberg, 1970; Heldt & Pfaff, 1969), the ADP,, and the ATP,, participating in thesereactions consist of the total intramitochondrial pool of ADP and ATP. Though Vignais and coworkers (Vignais et al., 1973; Duke & Vignais, 1969) state that the intramitochondrial pool of adenine nucleotides is homogeneous, they demonstrated that the phosphorylation of intramitochondrial ADP and of externally added ADP occur independently. We reported evidence (Bertha & Out, 1973) that the intermediates between the separate components of a single enzyme complex containing respiratory chain, F1 and adenine nucleotide translocator are not in rapid equilibrium with the total intramitochondrial pool of those intermediates. We have now obtained further evidence for a compartmentation of the intramitochondrial adenine nucleotides by the use of the ‘irreversible’ inhibitors of the adenine nucleotide transport, bongkrekic acid and gummiferine. The latter inhibitor has been shown by Klingenberg et al. (1973) to be irreversible. In co-operation with Mr. K. Duin, we have found that [3H]bongkrekic acid bound to specific binding sites (0.17nmol *mg of protein-’) in rat liver mitochondria equilibrates only extremely slowly with added unlabelled bongkrekic acid and is not removed by serum albumin. Binding to the specific binding sites parallels inhibition of adenine nucleotide transport. The exchange of added [14C]ADP with the intramitochondrial adenine nucleotides shows in the presence of bongkrekic acid or gummiferine inhomogeneous kinetics, namely (1) a fast reaction that can be ascribed to the exchange between added adenine nucleotides and those in the inside ‘compartments’ that are not excluded by the inhibitor from direct equilibration with the medium, and (2) a slow reaction, possibly revealing intramitochondrial exchange between ‘compartments’ that are and those that are not in direct interaction with the external adenine nucleotides. The extent to which the first reaction labels the intramitochondrial adenine nucleotides is decreased by increasing inhibitor concentrations, the half time being hardly influenced. It can also be demonstrated that, during steady-state state 3 phosphorylation at O”C, after addition of [14C]ADP with high specific radioactivity, [14C]ATP appears outside without an appreciable lag time that should occur if the ADP transported inwards or the ATP transported outwards equilibrates with the intramitochondrial adenine nucleotides (Bertha & Out, 1973). However, the rate of equilibration of external with internal adenine nucleotides (3.2nmol.min-’ ‘mg of protein-’) is nearly as high as the rate of ATP synthesis (3.9nmol.min-’ *mg of protein-’). In Table 1, results are given of measurements of the transport of ADP into the mitochondria. It seems clear that when the mitochondria are incubated in a system that maintains a high energy pressure, the rate of adenine nucleotide transport is about half the rate under conditions of low energy pressure. In this respect it should be mentioned that both Souverijn et al. (1973) and Vignais et al. (1973) have shown that the maximal rate of ADP transport is dependent on the metabolic conditions.