Multi-functional 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.
Ohyama
Y
Y
Department of Biochemistry, Hiroshima University School of Dentistry.
Masumoto
O
O
Usui
E
E
Okuda
K
K
D000818:Animals; D001667:Binding, Competitive; D001974:Bromosuccinimide / Q000494:pharmacology; D053493:Cholestanetriol 26-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006900:Hydroxylation; D007700:Kinetics; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D011489:Protein Denaturation; D051381:Rats; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; D001974:Bromosuccinimide