Morohashi K, Yoshioka H, Gotoh O, Okada Y, Yamamoto K, Miyata T, Sogawa K, Fujii-Kuriyama Y, Omura T
Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
J Biochem. 1987 Sep;102(3):559-68. doi: 10.1093/oxfordjournals.jbchem.a122089.
cDNA clones of the mRNA for bovine adrenal cytochrome P-450(11 beta) were isolated. Sequence analysis of a 4 kb long cDNA revealed the primary structure of P-450(11 beta), which consisted of 503 amino acids (Mr: 57,924) and contained an extension peptide of 24 amino acids at the NH2-terminus of the mature P-450(11 beta). molecule. A bovine genomic DNA containing the 1st exon and its leader sequence of P-450(11 beta) gene was also isolated from a bovine gene library. Determination of the transcription initiation site by S1 nuclease analysis using the cloned genomic DNA confirmed that the methionine codon near the 5' side of the 4 kb long cDNA was the initiation codon. Comparisons of the primary structures among P-450(11 beta) and other forms of cytochrome P-450 including P-450(SCC) indicated that the two mitochondrial P-450s, P-450(11 beta) and P-450(SCC), were significantly different from microsomal forms of cytochrome P-450. The homology between P-450(11 beta) and P-450(SCC) was 36%, which is higher than the values between P-450(11 beta) and various microsomal P-450s. An alignment of P-450(11 beta) and P-450(SCC) to give maximum matching showed four highly conserved regions (C-1, C-2, C-3, and C-4). The homology values of these regions were 58-70%, considerably higher than the overall homology between these two mitochondrial P-450s. A putative heme binding site and a steroid binding site were located in the conserved regions. Hydropathy profiles of P-450(11 beta) and P-450(SCC) were very similar. A definite difference was noticed at the NH2-terminal portion between mitochondrial and microsomal types of P-450. Microsomal type of cytochrome P-450 had a hydrophobic sequence consisting of about 20 amino acids, whereas mitochondrial type had an extension peptide containing many positively changed amino acids.
分离出了牛肾上腺细胞色素P - 450(11β)mRNA的cDNA克隆。对一个4kb长的cDNA进行序列分析，揭示了P - 450(11β)的一级结构，它由503个氨基酸组成（分子量：57,924），并且在成熟P - 450(11β)分子的NH2末端含有一个24个氨基酸的延伸肽。还从牛基因文库中分离出了包含P - 450(11β)基因第1外显子及其前导序列的牛基因组DNA。使用克隆的基因组DNA通过S1核酸酶分析确定转录起始位点，证实4kb长cDNA 5'端附近的甲硫氨酸密码子是起始密码子。对P - 450(11β)与其他细胞色素P - 450形式（包括P - 450(SCC)）的一级结构进行比较表明，两种线粒体P - 450，即P - 450(11β)和P - 450(SCC)，与微粒体形式的细胞色素P - 450有显著差异。P - 450(11β)与P - 450(SCC)之间的同源性为36%，高于P - 450(11β)与各种微粒体P - 450之间的值。将P - 450(11β)和P - 450(SCC)进行最大匹配比对显示有四个高度保守区域（C - 1、C - 2、C - 3和C - 4）。这些区域的同源性值为58 - 70%，大大高于这两种线粒体P - 450之间的总体同源性。一个假定的血红素结合位点和一个类固醇结合位点位于保守区域。P - 450(11β)和P - 450(SCC)的亲水性图谱非常相似。在线粒体和微粒体类型的P - 450的NH2末端部分注意到一个明显差异。微粒体类型的细胞色素P - 450有一个由约20个氨基酸组成的疏水序列，而线粒体类型有一个含有许多带正电荷氨基酸的延伸肽。
