MULTIFUNCTIONAL PROPERTY OF RAT-LIVER MITOCHONDRIAL CYTOCHROME-P-450

To solve the problem of whether a common enzyme catalyzes both 5-beta-cholestane-3-alpha,7-alpha, 12-alpha-triol 27-hydroxylation and 25-hydroxylation of 1-alpha-hydroxyvitamin D3 (a synthetic compound used therapeutically for vitamin D-deficient diseases) in rat liver mitochondria, enzymological and kinetic studies were performed. A cytochrome P-450 was purified from female rat liver mitochondria based on these catalytic activities and it was found that the two enzyme activities accompanied each other at all purification steps. The 5-beta-cholestane-3-alpha,7-alpha,12-alpha-triol 27-hydroxylation activity of the final preparation had a turnover number of 36 min-1, and the value of the corresponding 1-alpha-hydroxyvitamin D3 25-hydroxylation activity was 1.4 min-1. When the enzyme was partially denatured by heating at different temperatures, both enzyme activities declined in a parallel fashion. Treatment of the enzyme with N-bromosuccinimide decreased both enzyme activities in a similar manner. 5-beta-Cholestane-3-alpha,7-alpha,12-alpha-triol competitively inhibited 25-hydroxylation of 1-alpha-hydroxy-vitamin D3 and vice versa. From these results it was concluded that 5-beta-cholestane-3-alpha,7-alpha, 12-alpha-triol 27-hydroxylation and 1-alpha-hydroxyvitamin D3 25-hydroxylation are catalyzed by a common enzyme in rat liver mitochondria.