Klotz A V, Stegeman J J, Woodin B R, Snowberger E A, Thomas P E, Walsh C
Arch Biochem Biophys. 1986 Sep;249(2):326-38. doi: 10.1016/0003-9861(86)90009-3.
Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.
从海洋鱼类条纹锯鲷（尖口鲷）的肝脏微粒体中纯化出了两种新的细胞色素P-450形式。细胞色素P-450A（Mr = 52.5K）的一氧化碳结合型还原差光谱的最大吸收峰在447.5nm处，重组后具有适度的苯并[a]芘羟化酶活性（0.16 nmol/分钟/ nmol P-450）和乙氧基香豆素O-脱乙基酶活性（0.42 nmol/分钟/ nmol P-450）。在最佳条件下重组的细胞色素P-450A几乎只在6β位催化睾酮的羟化反应（0.8 nmol/分钟/ nmol P-450），并且还催化雌二醇的2-羟化反应。细胞色素P-450A对类固醇底物具有活性，我们认为它是微粒体睾酮6β-羟化酶活性的主要贡献者。细胞色素P-450A需要同种（条纹锯鲷）的NADPH-细胞色素P-450还原酶，并且所有检测的重组活性都因添加纯化的条纹锯鲷细胞色素b5而受到刺激。细胞色素P-450B（Mr = 45.9K）的一氧化碳结合型还原差光谱的最大吸收峰在449.5nm处，并且重组催化活性的速率较低。然而，细胞色素P-450B在几个不同位点氧化睾酮，包括15α位（0.07 nmol/分钟/ nmol P-450）。根据光谱性质、底物谱、在十二烷基硫酸钠-聚丙烯酰胺凝胶上的最小分子量、肽图谱以及与针对细胞色素P-450E产生的抗体缺乏交叉反应等标准，细胞色素P-450A和P-450B都与主要的苯并[a]芘羟化形式细胞色素P-450E不同。细胞色素P-450E与大鼠细胞色素P-450c共有表位，表明它是等效的酶，但条纹锯鲷细胞色素P-450A或P-450B与其他脊椎动物群体中已知的P-450同工酶之间可能存在的同源性尚不确定，尽管存在功能类似物。
