Rohde M, Mayer F, Meyer O
J Biol Chem. 1984 Dec 10;259(23):14788-92.
The localization of carbon monoxide oxidase (CO oxidase), the key enzyme in CO metabolism of Pseudomonas carboxydovorans, was examined using modified immunoferritin and protein A-gold techniques. Cell extracts were incubated with specific immunoglobulin G antibodies raised against CO oxidase, followed by treatment with ferritin-conjugated goat-anti-rabbit immunoglobulin G antibodies (pre-embedding labeling). Electron microscopic examination of ultrathin sections showed cytoplasmic membranes and inside-out vesicles labeled at the inner aspect, whereas the outer sides of protoplasts and membrane vesicles remained completely unlabeled. The highly sensitive protein A-gold method has been modified to allow labeling of CO oxidase with good ultrastructural preservation of the bacterial cell. Glutaraldehyde-fixed cells of P. carboxydovorans were osmificated and embedded in glycol methacrylate. Etched ultrathin sections were treated with sodium metaperiodate and incubated with the specific antibodies against CO oxidase. These antibodies were then allowed to react with protein A-gold complexes (postembedding labeling). Exponentially grown cells showed 87% of CO oxidase associated with the cytoplasmic membrane and 13% of the enzyme in the cytoplasm. The results indicate that CO oxidase is attached in vivo to the inner aspect of the cytoplasmic membrane and suggest interaction of the enzyme with a membrane-bound electron acceptor. The ratio of enzyme associated with the cytoplasmic membrane decreased to 50% in the stationary growth phase.
利用改良的免疫铁蛋白和蛋白A-金技术，对食羧基假单胞菌一氧化碳代谢中的关键酶——一氧化碳氧化酶（CO氧化酶）进行了定位研究。将细胞提取物与针对CO氧化酶产生的特异性免疫球蛋白G抗体孵育，随后用铁蛋白偶联的山羊抗兔免疫球蛋白G抗体处理（包埋前标记）。超薄切片的电子显微镜检查显示，细胞质膜和内翻囊泡在内侧被标记，而原生质体和膜囊泡的外侧则完全未被标记。对高度敏感的蛋白A-金方法进行了改良，以便在细菌细胞超微结构良好保存的情况下对CO氧化酶进行标记。将戊二醛固定的食羧基假单胞菌细胞进行渗透处理，并包埋在甲基丙烯酸乙二醇酯中。蚀刻的超薄切片用过碘酸钠处理，然后与针对CO氧化酶的特异性抗体孵育。然后让这些抗体与蛋白A-金复合物反应（包埋后标记）。指数生长期的细胞显示，87%的CO氧化酶与细胞质膜相关，13%的酶存在于细胞质中。结果表明，CO氧化酶在体内附着于细胞质膜的内侧，并提示该酶与膜结合电子受体存在相互作用。在稳定生长期，与细胞质膜相关的酶的比例降至50%。
