Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

Abstract

1. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside. 2. 2. [ 35S]carboxyatractyloside and [ 3H]bongkrekic acid bind to potato mitochondria with high affinity ( K d = 10 to 20 nM, n = 0.6–0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [ 3H]atractyloside binds to potato mitochondria with a much lower affinity ( K d = 0.45 μM) than carboxyatractyloside or bongkrekic acid. 3. 3. Bound [ 3H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [ 35S]carboxyatractyloside by bongkrekic acid and of bound [ 3H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP. 4. 4. Bongkrekic acid competes with [ 35S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [ 35S]carboxyactratyloside binding. 5. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.