Studies of adrenal steroid hydroxylation

Purification and optical studies of cytochrome P‐450 from bovine adrenocortical microsomes

“…Recently, two different kinds ofmitochondrialP-450 cytochromes, catalyzing the cholesterol side-chain cleavage (P-450scc) and the steroid 11/3-hydroxylation (P-4501U3) have been highly purified from bovine adrenal cortex [1][2][3]. Investigations on the reaction mechanism of cytochrome P-450 in adrenal endoplasmic reticulum have been carried out using a microsomal particle or its extract [4][5][6][7][8][9][10][11][12][13]. However, so far as we know, no isolation of cytochrome P-450 from the microsomes has been achieved.…”

Biological Aspects of Cytochrome P450 and Associated Hydroxylation Reactions

“…The presence of cytochrome P450 could not be demonstrated in active 21-hydroxylase of 17a-hydroxyprogesterone and the examination of factors inhibiting the reaction could support this possibility. However, Mackler et al [92], having achieved partial separation of the 21-hydroxylation of progesterone and 17a-hydroxyprogesterone, considered that one enzyme system effected these hydroxylations with possibly separate sites of binding of the steroids to the enzyme or partially divergent pathways with separate cofactors for the individual hydroxylations. The latter possi bility accords well with the observation that the pattern of hydroxylation products of progesterone depends on the concentration of NADPH employ ed [135] and that the Km for NADPH in the 21-hydroxylation of progesterone is 10.4 fj.mol/1 while that in the 21-hydroxylation of 17a-hydroxyprogesterone is 0.6 [imol/1 [152], While these indications suggest the possible existence of isoenzymic forms of the flavoprotein, NADPH-cytochrome P450 reductase, it may be that the NADPH-binding sites of otherwise simi lar flavoproteins are modified by interaction of the flavoprotein with different species of cytochrome P450.…”

“…Further experimental observations on the steroid 21-hydroxylation reactions of human adrenocortical microsomes are presented elsewhere [153]. hydroxylation with separate cofactor-binding sites for the different hydrox ylations [92]. Scheme B considers the theoretical possibility that the second reducing equivalent required for the hydroxylation reaction is obtained by cytochrome P450 directly.…”