Immunochemical evidence of trifluoroacetylated cytochrome P-450 in the liver of halothane-treated rats.
Immunochemical evidence of trifluoroacetylated cytochrome P-450 in the liver of halothane-treated rats.
复制标题
氟烷治疗大鼠肝脏中三氟乙酰化细胞色素 P-450 的免疫化学证据。
作者：
H. Satoh;J. Gillette;H. Davies;R. Schulick;L. Pohl
给苯巴比妥处理的大鼠注射氟烷4h后，分离肝脏亚细胞部分，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离蛋白，转移到硝酸纤维素膜上，用抗三氟乙酰化抗体进行免疫化学染色。微体部分含有最高水平的三氟乙酰化加合物。其主要的三氟乙酰化组分经免疫化学鉴定为苯巴比妥诱导的细胞色素P-450(54 KDa)，而其他观察到的三氟乙酰化蛋白组分(59 KDa)未被鉴定。质膜部分还含有一个54 kDa的三氟乙酰化加合物，该加合物与54 kDa细胞色素P-450免疫化学相关。接受氟烷治疗的未治疗大鼠的微粒体只含有59 kDa的三氟乙酰化蛋白质部分。对氟烷结合氧化代谢产物的免疫化学染色的特异性得到证实，这一发现表明，用重氢氟烷处理的大鼠比用氟烷处理的大鼠的肝脏蛋白染色要少得多。这些结果表明，氟烷的CF3COX氧化代谢产物是如此活跃，以至于它主要与产生它的细胞色素P-450结合。
Four hours after the administration of halothane to phenobarbital-pretreated rats, subcellular fractions of liver were isolated and the proteins in the fractions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and immunochemically stained with anti-trifluoroacetylated antibodies. The microsomal fraction contained the highest level of trifluoroacetylated adducts. Its major trifluoroacetylated component was immunochemically identified as a phenobarbital-inducible form of cytochrome P-450 (54 kDa), whereas the other observed trifluoroacetylated protein fraction (59 kDa) was not identified. The plasma membrane fraction also contained a 54-kDa trifluoroacetylated adduct, which was immunochemically related to the 54-kDa cytochrome P-450. Microsomes from untreated rats that were administered halothane contained only the 59-kDa trifluoroacetylated protein fraction. The specificity of the immunochemical staining for the bound oxidative metabolite of halothane was confirmed by the finding that rats treated with deuterated halothane had considerably less stained liver proteins than did those treated with halothane. These results suggest that the CF3COX oxidative metabolite of halothane is so reactive that it binds predominantly to the cytochrome P-450 that produced it.