Further evidence that there is more than one adrenal 21-hydroxylase system

Abstract

The 21-hydroxylase activity of microsomes isolated from bovine adrenal cortex have been assayed using [21- 3H]17-hydroxypregnenolone and [1,2- 3H]17-hydroxyprogesterone as substrates. When the assays are performed in the presence of an NADH regenerating system, to inhibit steroid 3β-hydroxy isomerase-dehydrogenase activity, the microsomes oxidize the 3β-hydroxy-5-ene steroid at a rate of 0.37nmol/min·nmol cytochrome P-450 and the 3-keto-4-ene steroid at a rate of 6.4 nmol/min · nmol. When the microsomes are solubilized with Triton CF-54 they lose the ability to oxidize the 3-hydroxy-5-ene steroid, while the specific activity of the microsomes for the 3-keto-4-ene steroid is enhanced 3-fold. In contrast, when the microsomes are solubilized with sodium cholate, their specific activity towards the 4-ene steroid is decreased by 50% while the specific activity for a low concentration of the 5-ene steroid, 1 μM, is unchanged. In addition, when the oxidations of the labeled steroids (at 1 μM) by the microsomes, are examined in the presence of unlabeled 17-hydroxyprogesterone (at 20 μM) the oxidation of the 3-keto-4-ene steroid is inhibited by 92% while the oxidation of the 3β-hydroxy-5-ene steroid is only inhibited by 20%. These results all suggest that there are at least two 21-hydroxylases in bovine adrenal tissue, one of which can utilize the 3-keto-4-ene steroids only, the other of which, in addition, can utilize the 3β-hydroxy-5-ene steroids as substrates.