Purification and characterization of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase from female rat liver mitochondria.

5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase (5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, NADPH:oxygen oxidoreductase (26-hydroxylating), EC 1.14.13.15) was purified from female rat liver mitochondria based on its catalytic activity. The final preparation of the enzyme showed a single major band on the sodium dodecyl sulfate-polyacrylamide gel electrophoretogram. The content of purified enzyme was 12 nmol/mg of protein, and the specific activity was 431 nmol/min/mg of protein. The molecular weight of the enzyme was determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 52,500. The absorption spectra of the purified enzyme and that of the dithionite-reduced CO complex showed peaks at 417 and 450 nm, respectively, indicating the enzyme belongs to the cytochrome P-450 family. Upon reconstitution with the electron-transferring system of the adrenal (adrenodoxin and NADPH-adrenodoxin reductase), the enzyme showed high activity hydroxylating 5 beta-cholestane-3 alpha,7 alpha-12-triol at position 27 with a turnover number of 35.5 min-1 and Km of 6.3 microM. The enzyme activity was completely lost when the electron-transferring system was replaced by that of microsomes (NADPH-cytochrome P-450 reductase purified from rat liver microsomes), confirming that the P-450 enzyme was of the mitochondrial type, but not of the microsomal. The omission of cytochrome P-450, adrenodoxin, or NADPH-adrenodoxin reductase resulted in complete loss of enzyme activity. The specific activity toward 5 beta-cholestane-3 alpha, 7 alpha-diol was less than one-half that toward cholestanetriol and that toward cholesterol was about one-fiftieth. The enzyme showed no activity toward xenobiotics such as benzphetamine, 7-ethoxycoumarin, and benzo[a]pyrene. Its activity was not inhibited by metyrapone and slightly inhibited by aminoglutethimide. The enzyme activity was markedly lowered in an atmosphere of CO/O2/N2, 40/20/40.