Haass-Männle H, Zimmermann H W
Institut für Physikalische Chemie der Universität Freiburg, Freiburg, Germany.
J Photochem Photobiol B. 1997 Nov;41(1-2):90-102. doi: 10.1016/s1011-1344(97)00088-2.
The lipophilic, cationic fluorochrome azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living yeast cells (Saccharomyces cerevisiae WT X 2180). It contains a photosensitive diazirine ring and is suitable for photoaffinity labelling. By combining photoaffinity labelling, micro-gel electrophoresis (SDS-PAGE), and detection of the APMC fluorescence with a microfluorimeter, we established a highly sensitive procedure for determining the apparent molecular weight of the APMC-labelled proteins in yeast cells. On vital staining at 0.1 microM APMC for 30 min, only one mitochondrial protein with an apparent molecular weight of 40 kDa is labelled with high intensity. At increased dye concentrations proteins of 47 and 49 kDa are labelled too, however not until all binding sites of the 40 kDa protein are occupied. Obviously, the APMC cations have a pronounced affinity for this protein. It was shown by fractional centrifugation that the labelled 40 kDa protein is a constituent of the inner mitochondrial membrane. One driving force for the accumulation of the APMC cations is the trans-membrane potential (TMP) across the inner mitochondrial membrane. Consequently, uncouplers like dinitrophenol (DNP) and carbonylcyanidechlorophenyl-hydrazone (CCCP), ionophores (valinomycin, gramicidin), and inhibitors of the respiratory chain (myxothiazol, KCN), which decrease the TMP, also diminish the APMC accumulation and labelling. And conversely, drugs, which hyperpolarize the inner membrane (nigericin, atractyloside), favour APMC labelling. Another driving force of APMC accumulation is the dye's lipophilicity, which facilitates dye accumulation by hydrophobic interaction with the very lipophilic proteins of the inner mitochondrial membranes. This was shown by competitive double staining experiments. Thiamine strongly inhibits APMC labelling. Obviously, the transport of the APMC cations is facilitated by the thiamine carrier, and thiamine competes for the same binding sites, which are occupied by the dye cations. Chloramphenicol is an inhibitor of the mitochondrial protein synthesis without affecting the TMP. On preincubation, chloramphenicol completely quenches the signal of the 40 kDa protein. Therefore, this protein must be encoded on the mtDNA. The only 40 kDa protein with adequate properties is the subunit I of cytochrome c oxidase. Obviously, it is the preferred target of the APMC cations on photoaffinity labelling. This assignment agrees with the strong hydrophobicity of the labelled 40 kDa protein, which was tested with various detergents. It also agrees with the solvatochromism of the protein-bound APMC label, and finally with the paralellism of the labelled protein with cytochrome c oxidase on fractional ammonium sulfate precipitation.
亲脂性阳离子荧光染料偶氮戊基甲基吲哚碳菁（APMC）可特异性地对活酵母细胞（酿酒酵母野生型X2180）中的线粒体进行染色。它含有一个光敏重氮环，适用于光亲和标记。通过结合光亲和标记、微量凝胶电泳（SDS-PAGE）以及用微量荧光计检测APMC荧光，我们建立了一种高度灵敏的方法来测定酵母细胞中APMC标记蛋白的表观分子量。用0.1微摩尔/升的APMC进行30分钟的活体染色时，只有一种表观分子量为40 kDa的线粒体蛋白被高强度标记。当染料浓度增加时，47 kDa和49 kDa的蛋白也会被标记，但直到40 kDa蛋白的所有结合位点都被占据才会出现这种情况。显然，APMC阳离子对这种蛋白具有显著的亲和力。分级离心表明，被标记的40 kDa蛋白是线粒体内膜的一个组成部分。APMC阳离子积累的一个驱动力是线粒体内膜两侧的跨膜电位（TMP）。因此，像二硝基苯酚（DNP）和羰基氰化物氯苯腙（CCCP）这样的解偶联剂、离子载体（缬氨霉素、短杆菌肽）以及呼吸链抑制剂（粘噻唑、KCN），它们会降低TMP，也会减少APMC的积累和标记。相反，使内膜超极化的药物（尼日利亚菌素、苍术苷）则有利于APMC标记。APMC积累的另一个驱动力是染料的亲脂性，这通过与线粒体内膜非常亲脂的蛋白进行疏水相互作用促进了染料的积累。这在竞争性双重染色实验中得到了证明。硫胺素强烈抑制APMC标记。显然，硫胺素载体促进了APMC阳离子的转运，并且硫胺素竞争与染料阳离子占据的相同结合位点。氯霉素是线粒体蛋白质合成的抑制剂，不影响TMP。预孵育时，氯霉素会完全淬灭40 kDa蛋白的信号。因此，这种蛋白一定是由线粒体DNA编码的。唯一具有适当性质的40 kDa蛋白是细胞色素c氧化酶的亚基I。显然，它是光亲和标记时APMC阳离子的首选靶点。这一归属与被标记的40 kDa蛋白的强疏水性相符，该疏水性已用各种去污剂进行了测试。它也与蛋白结合的APMC标记的溶剂化显色相符，最后与分级硫酸铵沉淀时被标记蛋白与细胞色素c氧化酶的平行性相符。
