Identification and localization of amino acid substitutions between two phenobarbital-inducible rat hepatic microsomal cytochromes P-450 by micro sequence analyses.
Identification and localization of amino acid substitutions between two phenobarbital-inducible rat hepatic microsomal cytochromes P-450 by micro sequence analyses.
复制标题
通过微序列分析鉴定和定位两种苯巴比妥诱导的大鼠肝微粒体细胞色素 P-450 之间的氨基酸取代。
DOI：
10.1073/pnas.80.5.1169
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发表时间：
1983
影响因子：
11.1
通讯作者：
J. Shively
中科院分区：
文献类型：
--
作者：
P. Yuan;D. Ryan;W. Levin;J. Shively
通过对大鼠肝微粒体细胞色素P-450--P-450B和P-450E--的NH2末端和胰酶片段的微序列分析，比较了两种同工酶的异同。这两个苯巴比妥诱导的血红蛋白在免疫化学上与细胞色素P-450B抗体难以区分，具有广泛的序列同源性。对天然蛋白质的自动埃德曼降解显示，前35个残基的氨基酸相同。到目前为止，对占每个蛋白质分子约75%的胰蛋白酶多肽的序列测定表明，这两种同工酶之间存在10个氨基酸差异。氨基酸序列分析结果表明，Fujii-Kuriyama等人报道了其中两个cDNA，即PCP-450pb1和PCP-450pb4。访问数/每百万人：Reach of[Fujii-Kuriyama，Y.，Mizukami，Y.，Kamajiri，K.，Sogawa，K.&Muramatsu，M.(1982)Proc.娜塔莉。阿卡德。SCI。美国79,2793-2797]编码细胞色素P-450b，而编码细胞色素P-450e的第三个cDNAPCP-450pb2的核苷酸序列与另外两个略有不同(6个氨基酸替换)。除了确定这些克隆的cDNA的同源性外，我们还提供了细胞色素P-450b和P-450e之间另外七个氨基酸差异的直接证据，这些差异发生在克隆的细胞色素P-450e的cDNA编码的区域(Arg358)之外。通过微序列分析和重组DNA技术测定的氨基酸序列共同揭示了这两种同工酶之间的13个氨基酸差异。这份报告强调了两种不同的分子方法的互补性，以阐明具有广泛结构同源性的同工酶的氨基酸序列。
Two isozymes of rat liver microsomal cytochrome P-450--P-450b and P-450e--were compared by micro sequence analyses of their NH2 termini and tryptic fragments. These two phenobarbital-inducible hemoproteins, which are immunochemically indistinguishable with antibody against cytochrome P-450b, have extensive sequence homology. Automated Edman degradation of the native proteins revealed identical amino acids for the first 35 residues. Sequence determinations of the tryptic peptides, which constitute approximately 75% of each protein molecule, have thus far shown 10 amino acid differences between the two isozymes. Results of our amino acid sequence analyses established that two of the cDNAs, pcP-450pb1 and pcP-450pb4, reported by Fujii-Kuriyama et al. [Fujii-Kuriyama, Y., Mizukami, Y., Kamajiri, K., Sogawa, K. & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. USA 79, 2793-2797] encode cytochrome P-450b whereas pcP-450pb2, a third cDNA whose nucleotide sequence differed slightly from that of the other two (six amino acid substitutions), encodes cytochrome P-450e. In addition to establishing the identity of these cloned cDNAs we provide direct evidence for seven additional amino acid differences between cytochromes P-450b and P-450e that occur beyond the region (Arg358) encoded by the cloned cDNA for cytochrome P-450e. Together, the amino acid sequences determined by micro sequence analysis and recombinant DNA techniques reveal 13 amino acid differences between these two isozymes. This report highlights the complementary nature of two different molecular approaches to elucidation of the amino acid sequences of isozymes with extensive structural homology.