Heinemann F S, Ozols J
J Biol Chem. 1982 Dec 25;257(24):14988-99.
The phenobarbital-induced isozyme of cytochrome P-450 was isolated from rabbit liver microsomes and subjected to chemical and proteolytic degradation. The resulting peptides were isolated exclusively by reverse phase high pressure liquid chromatography. Automated sequence analysis was employed to determine the linear arrangement of 385 residues and the order of all CNBr peptides. The NH2 terminus of the cytochrome P-450 consists of five hydrophobic segments 18 to 23 residues long, separated by polar regions of 8 to 15 residues. The COOH terminus contains a hydrophilic region 120 residues long as well as several hydrophobic segments. A tridecapeptide, previously found to be analogous to a tryptic peptide from a constitutive P-450 isozyme (Ozols, J., Heinemann, F. S., and Johnson, E. F. (1981) J. Biol. Chem. 256, 11405-11408) was positioned within the hydrophilic region of the COOH terminus. The amino acid sequence is interpreted in terms of a model for the topology of the cytochrome in the membrane which can be used as a basis for further experiments. Comparison of this sequence with the cytochrome P-450CAM sequence (Haniu, M., Armes, L. G., Tanaka, M., Yasunobo, K. T., Shastry, B. S., Wagner, G. C., and Gunsalus, I. C. (1982) Biochem. Biophys. Res. Commun. 105, 889-894) revealed a single homologous region which contains a cysteinyl residue. The amino acid sequence of his segment is as follows: P-450 microsomal/P-450CAM:Ile/Leu-Cys-Leu-Gly-Gln/Glu-Ser/Gly-Leu/Ile-Ala-Arg. The amino acid sequence data were also compared with the amino acid sequence deduced from the nucleotide sequence of phenobarbital-inducible cytochrome P-450 mRNA from rat liver (Kurijama-Fuji, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). This comparison suggests that the structure of the phenobarbital-induced isozyme is highly conserved among mammalian species.
从兔肝微粒体中分离出苯巴比妥诱导的细胞色素P - 450同工酶，并对其进行化学和蛋白酶降解。所得肽段仅通过反相高压液相色谱法分离。采用自动序列分析来确定385个残基的线性排列以及所有溴化氰肽段的顺序。细胞色素P - 450的氨基末端由五个长度为18至23个残基的疏水片段组成，被8至15个残基的极性区域隔开。羧基末端包含一个120个残基长的亲水区域以及几个疏水片段。先前发现的一个十三肽与组成型P - 450同工酶的胰蛋白酶肽段类似（奥佐尔斯，J.，海涅曼，F. S.，和约翰逊，E. F.（1981年）《生物化学杂志》256卷，11405 - 11408页），位于羧基末端的亲水区域内。根据膜中细胞色素拓扑结构模型对氨基酸序列进行了解释，该模型可作为进一步实验的基础。将此序列与细胞色素P - 450CAM序列（羽仁，M.，阿姆斯，L. G.，田中，M.，安野保男，K. T.，沙斯特里，B. S.，瓦格纳，G. C.，和冈萨卢斯，I. C.（1982年）《生物化学与生物物理学研究通讯》105卷，889 - 894页）进行比较，发现了一个单一的同源区域，其中包含一个半胱氨酰残基。该片段的氨基酸序列如下：P - 450微粒体/P - 450CAM：异亮氨酸/亮氨酸 - 半胱氨酸 - 亮氨酸 - 甘氨酸 - 谷氨酰胺/谷氨酸 - 丝氨酸/甘氨酸 - 亮氨酸/异亮氨酸 - 丙氨酸 - 精氨酸。还将氨基酸序列数据与从大鼠肝苯巴比妥诱导型细胞色素P - 450 mRNA的核苷酸序列推导的氨基酸序列进行了比较（栗岛富士，Y.，水神，Y.，川尻，K.，曾川，K.，村松，M.（1982年）《美国国家科学院院刊》79卷，2793 - 2797页）。这种比较表明，苯巴比妥诱导的同工酶结构在哺乳动物物种中高度保守。
