Ziegler M, Penefsky H S
Department of Biochemistry and Molecular Biology, State University of New York, Health Science Center, Syracuse 13210.
J Biol Chem. 1993 Dec 5;268(34):25320-8.
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.
将荧光性、非渗透性的pH指示剂吡喃黄素掺入亚线粒体颗粒（吡喃黄素 - SMP）中，可监测由于呼吸链底物氧化或ATP水解导致质子转运所引起的囊泡内pH变化。向牛肉心吡喃黄素 - SMP中添加寡霉素后，吡喃黄素荧光出现与pH无关的猝灭。猝灭受到亚线粒体颗粒内外腺嘌呤核苷酸的影响。猝灭所需核苷酸的性质类似于腺嘌呤核苷酸转运酶而非F1 - ATP酶的特异性。用尿素处理颗粒从吡喃黄素 - SMP中去除F1，并不会改变寡霉素诱导的猝灭。当将腺嘌呤核苷酸转运酶的特异性抑制剂苍术苷与吡喃黄素共同掺入亚线粒体颗粒时，可防止寡霉素诱导的猝灭。分别在添加寡霉素之前或之后向吡喃黄素 - SMP中添加邦克雷酸，可防止或逆转寡霉素依赖性猝灭，但前提是颗粒内存在ATP。酿酒酵母的一个缺乏转运酶基因的突变体表现出寡霉素依赖性荧光猝灭，且不受邦克雷酸抑制。这些结果支持这样一种解释，即寡霉素促进荧光探针在亚线粒体颗粒的一个区域（可能是F0F1复合物）中隔离，从而导致荧光猝灭。观察到的猝灭可以通过一种方式进行调节，这表明转运酶与F0之间存在相互作用。
