Ohgiya N, Yokota H, Yuasa A
Department of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido.
J Biochem. 1989 Feb;105(2):234-8. doi: 10.1093/oxfordjournals.jbchem.a122645.
A form of cytochrome P-450 generally catalyzing benzo[a]pyrene (B[a]P) hydroxylation was purified from liver microsomes of untreated rats on the basis of the catalytic activity. The purification procedures consisted of cholate solubilization and chromatography in 3 steps, on DEAE-Toyopearl (at room temperature), hydroxylapatite, and CM-Toyopearl columns. Cytochrome P-450 purified in this way (named P-450/B[a]P) was homogeneous on SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated to be 51,000. The absorption spectra of the oxidized form of P-450/B[a]P showed a Soret peak at 417 nm, characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 451 nm. Immunochemical analysis of P-450/B[a]P indicated that P-450/B[a]P is immunologically distinct from P-450b (a major phenobarbital-inducible form of P-450) and P-450c (a major 3-methylcholanthrene-inducible form of P-450, which highly catalyzes the hydroxylation of B[a]P). B[a]P hydroxylase activity in liver microsomes of untreated rats was inhibited to about 20% by the P-450/B[a]P antibody. These results demonstrate that P-450/B[a]P is a different form of P-450 from P-450b and P-450c, and generally catalyzes B[a]P hydroxylation in liver microsomes of untreated rats.
基于催化活性，从未经处理的大鼠肝脏微粒体中纯化出一种通常催化苯并[a]芘（B[a]P）羟基化的细胞色素P-450形式。纯化步骤包括用胆酸盐增溶并通过三步色谱法，即分别在DEAE- Toyopearl柱（室温下）、羟基磷灰石柱和CM-Toyopearl柱上进行色谱分离。以这种方式纯化得到的细胞色素P-450（命名为P-450/B[a]P）在SDS-聚丙烯酰胺凝胶电泳上呈均一性，估计分子量为51,000。P-450/B[a]P氧化形式的吸收光谱在417nm处显示一个Soret峰，这是低自旋血红蛋白的特征，而还原型细胞色素P-450-CO复合物的Soret峰在451nm处。对P-450/B[a]P的免疫化学分析表明，P-450/B[a]P在免疫上与P-450b（P-450的主要苯巴比妥诱导形式）和P-450c（P-450的主要3-甲基胆蒽诱导形式，其高度催化B[a]P的羟基化）不同。未经处理的大鼠肝脏微粒体中的B[a]P羟化酶活性被P-450/B[a]P抗体抑制至约20%。这些结果表明，P-450/B[a]P是一种与P-450b和P-450c不同的P-450形式，并且通常在未经处理的大鼠肝脏微粒体中催化B[a]P的羟基化。
