Some principle effects of bongkrekic acid on the binding of adenine nucleotides to mitochondrial membranes.

Abstract

The effect of bongkrekic acid on the binding of ADP to mitochondrial membranes is characterized and studied in relation to a number of parameters. Bongkrekic acid increases the binding of ADP to mitochondrial membranes (from beef heart, rat heart and rat liver) in contrast to atractyloside. The “specific” ADP binding (i.e. binding at the carrier sites), under the influence of bongkrekic acid, is differentiated by the combined application of bongkrekic acid and atractyloside so that the unspecific, non‐carrier‐linked binding portions are substracted. The binding increase by bongkrekic acid is specific both for ADP and ATP and excludes AMP and other nucleotides such as IDP etc. The dependence of the binding on the concentration of ADP shows that under the influence of bongkrekic acid the affinity for ADP is increased by about 25‐ to 100‐fold, yielding a dissociation constant KD of 0.2 to 0.02 μM. The dissociation rate of bound ADP in the presence of bongkrekic acid as measured by the exchangeability of bound [14C]ADP against ADP decreases more than 200‐fold. The removal of [14C]ADP by atractyloside is even fully inhibited by bongkrekic acid. The inhibition by bongkrekic acid of the ADP dissociation from the carrier explains the observed inhibition by bongkrekic acid of the overall ADP translocation. The specific ADP binding is smaller when bongkrekic acid is added before than after ADP. The binding sites, differentiated by the sequence of bongkrekic acid addition, are interpreted to reflect inner binding sites which can be reached by bongkrekic acid due to its lipophilic nature. In certain cases this difference is of equal size to the portion of high affinity sites for ADP without bongkrekic acid which previously also had been explained to represent the inner sites exposed to endogenous adenine nucleotides. With double labelling the binding of endogenous adenine nucleotides can be directly shown to be increased during preincubation with bongkrekic acid causing an apparent decrease in binding of exogenous ADP. Thus at high concentration of bongkrekic acid all sites can be blocked by endogenous adenine nucleotides. The additional binding of ADP by bongkrekic acid is remarkably slow depending on several parameters (e.g. half‐time, t1/2, ranging from 0.5 to 5 min at pH 7.0, 20°C). Preincubation with bongkrekic acid only partially eliminates the slow time course, indicating that transition of sites to high affinity requires simultaneous presence of bongkrekic acid and ADP. The influence of bongkrekic acid on the binding of ADP is strongly temperature‐dependent. The temperature effect is of kinetic nature since once applied at increased temperature, bongkrekic acid remains effective in the ADP binding at lower temperature. The temperature effect is diminished at lower pH and at high concentration of bongkrekic acid. The bongkrekic acid effect on ADP binding is strongly pH‐dependent. It is effective only at pH < 7.2 and completely abolished at pH > 7.6. The pK of the bongkrekic acid effect is independent of the concentration of ADP but is increased with the concentration of bongkrekic acid. Two theories are discussed: (a) a cooperative increase of the binding for ADP by bongkrekic acid, i.e., existence of substrate and regulatory sites, or (b) binding of bongkrekic acid to the substrate site of the carrier, however, preferentially when located on the inner membrane surface, resulting in a redistribution of all carrier sites to the inner surface. Thus the function of a mobile carrier would be demonstrated on the molecular level.