[11-beta-Steroid hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria: isolation, physico-chemical...

It was shown that of the two cytochromes P-450, i.e. cholesterol-hydroxylating (SCC) and 11 beta-steroid-hydroxylating (11 beta) ones, involved in mitochondrial steroidogenesis in adrenal cortex cells cytochrome P-450 (11 beta) is more tightly bound to the membrane. The quantitative ratio of P-450 (SCC)/P-450 (11 beta) in the mitochondrial membrane is 2 to 1. The isolation procedure for cytochrome P-450 (11 beta) included separation of cytochromes P-450 (11 beta) and P-450 (SCC) by ammonium sulfate fractionation in the presence of sodium cholate, Tween 80 and affinity chromatography on adrenodoxin-Sepharose allowing to achieve a high degree of cytochrome P-450 (11 beta) purification. The obtained protein has a high specific heme content (19 nmole per mg of protein), minimal molecular weight (47000), is homogeneous as can be evidenced by Na-DS polyacrylamide gel electrophoresis and N-terminal analysis, possesses spectral properties typical for a high spin hemoprotein and is highly active in the reaction of steroid substrate hydroxylation. The Km values for hydroxylation of deoxycorticosterone (DOC), 11-deoxycorticol and testosterone are 0.69, 0.54 and 0.21 s-1, respectively. The use of adrenodoxin possessing a high stabilizing action allows to obtain cytochrome P-450 (11 beta) free from substrate (a low spin form) by washing of adrenodoxin-Sepharose adsorbed hemoprotein with large volumes of steroid-free phosphate buffer. The eluted cytochrome P-450 (11 beta) reveals spectral properties typical for the low spin hemoprotein; an addition of DOC induces type I spectral changes. The Kd values for the cytochrome P-450 (11 beta)-steroid complex determined by spectrophotometric titration at 10 degrees for DOC and testosterone are 0.32 and 0.67 muM, respectively.