Interaction of (3H) bongkrekic acid with the mitochondrial adenine nucleotide translocator

Chemical labeling by 3H and biosynthetic labeling by 14C of bongkrekic acid (BA) are described. In the rat liver cell, mitochondria are the only subcellular particles to bind [3H]BA with high affinity. The high affinity sites for BA in mitochondria are located in the inner membrane. High affinity binding sites for BA are only displayed at pH below 7; they amount to 0.15-0.20 nmol/mg of protein in rat liver mitochondria and to 1.1-1.3 nmol/mg of protein in rat heart mitochondria. These values are similar to those found for the high affinity atractyloside binding sites and for the carboxyatractyloside binding sites. The kinetic parameters for BA binding to rat heart mitochondria at 20 degrees C are Kd = 10-40 X 10(-9) M, k+1 = 0.7 X 10(5) M-1 s-1, k-1 = 1.4 X 10(-3) M s-1. Binding assays carried out with rat heart mitochondria, under equilibrium conditions, showed that the amount of BA bound to high affinity sites increases with temperature and reaches the maximum value of 1.1-1.3 nmol/mg of protein at 32-35 degrees C. At lower temperatures, and under equilibrium conditions, a significant fraction of high affinity sites remains masked and is not titrated by BA; these masked BA sites are revealed by addition of micromolar concentrations of ADP or by energization of the mitochondria. Carboxyatractyloside added to rat heart mitochondria preloaded with [3H]BA is able to displace part of the bound [3H]BA. Displacement of the bound BA is enhanced by simultaneous additions of carboxyatractyloside plus ADP, or by energization of the mitochondria. The synergistic effect of carboxyatractyloside and ADP on displacement of bound [3H]BA is also observed in isolated inner membrane vesicles from rat liver mitochondria. When BA is preincubated with rat heart mitochondria before addition of [14C]ADP for assay of ADP transport, the inhibition of ADP transport is a mixed-type inhibition. When BA is preincubated with the mitochondria together with a very small concentration of ADP (less than 0.5 muM), the inhibition of [14C]ADP transport is markedly increased (up to ten times) and it becomes typically uncompetitive, which suggests the formation of a ternary complex, carrier-ADP-BA. The transition from a mixed-type inhibition, with high Ki value, to an uncompetitive type of inhibition, with low Ki value, upon addition of ADP, is explained by an ADP-induced conformational change of the ADP translocator.