bongkrekic-acid and pyranine

Incorporation of the fluorescent, nonpermeant pH indicator pyranine into submitochondrial particles (pyranine-SMP) permitted monitoring of intravesicular pH changes brought about by proton translocation due to oxidation of respiratory chain substrates or to hydrolysis of ATP. Addition of oligomycin to beef heart pyranine-SMP was followed by a pH-independent quenching of pyranine fluorescence. Quenching was influenced by the presence of adenine nucleotides both inside and outside the submitochondrial particles. The nature of the nucleotides required for quenching resembled the specificity of the adenine nucleotide translocase rather than F1-ATPase. Removal of F1 from pyranine-SMP by treatment of the particles with urea did not alter oligomycin-induced quenching. Atractyloside, a specific inhibitor of the adenine nucleotide translocase, prevented oligomycin-induced quenching when the inhibitor was coincorporated into submitochondrial particles with pyranine. Bongkrekic acid prevented or reversed the oligomycin-dependent quenching when added to pyranine-SMP either before or after oligomycin, respectively, but only when ATP was present within the particles. A mutant of Saccharomyces cerevisiae, lacking translocase genes, exhibited oligomycin-dependent fluorescence quenching which was not inhibited by bongkrekic acid. The results support the interpretation that oligomycin promotes sequestration of the fluorescent probe in a region of the submitochondrial particle, probably the F0F1 complex, that leads to a quenching of fluorescence. The observed quenching can be modulated in a way that suggests an interaction between the translocase and F0.

Topics: Adenosine Triphosphate; Animals; Arylsulfonates; Atractyloside; Bongkrekic Acid; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cattle; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Oligomycins; Proton-Translocating ATPases; Ribonucleotides; Saccharomyces cerevisiae; Spectrometry, Fluorescence; Submitochondrial Particles; Substrate Specificity