微粒体细胞色素P-450、NADPH-细胞色素P-450还原酶的纯化与重组活性

经苯巴比妥钠诱导的雄性大白鼠的肝微粒体纯化的细胞色素P-450同功酶组份,经SDS-PAGE鉴定呈电泳纯,分子量为55kD。部分纯化的NADPH-细胞色素P-450还原酶,含72和77kD两个蛋白质组分。上述细胞色素P-450和NADPH-细胞色素P-450还原酶与卵磷脂制备的脂质体重组后的活性试验表明,对艾氏剂有环氧化作用,对环已烷有羟化作用,对溴氰菊酯的羟化作用微弱。当重组系统中缺少细胞色素P-450组份时,对环已烷不再起作用。同时还研究了纯化的细胞色素P-450的光谱特性。

Purification of Microsomal Cytochrome P-450, NADPH-Cyto-chrome P-450 Reductase from Phenobarbital-induced Rat Liver and Activities of the Reconstituted Enzyme System

A major form of cytochrome P-450 isozyme was purified from hepatic-microsomcs of sodium phenobarbital induced male rats by using a procedure involving PEG fractionation and chromatography on column of DEAE-sephacel (3.6**********x22cm) in the presence of 2% Lubrol-PX and eluted with potassium phosphate buffer.Only a single protein band of 55kD, which exhibited an absorption peak on the CO-differe-nce spectra at 450nm,was found on the 10% SDS-PAGE separation gel.NADPH-cyt-ochrome P-450 reductase was partially purified: with ammonium sulfate fractionation and chromatography on DEAE-sephacel (2.8**********X20cm)column containing 1.5% Lubrol-PX in Tris-HCl buffer.Two protein bands with molecular weight 72 and 77 kD respectively were found on the 7% SDS-PAGE separation gel.The reconstituted enzyme system composed of cytochrome P-450, NADPH-cytochrome P-450 reductase and ph-ospholipid was active in hydroxylation of cyclohexane and epoxidation of aldrin but acted poorly in hydroxylation of deltamethrin.