The purposes of this study were 1) to devise a method by which microsomal membranes couldbe prepared from homogeneous glomerulosa or fasciculata-reticularis cell populations from beef adrenals, and 2) to compare these zones with regard to C21-steroid 17α-hydroxylation of progesterone and 21-hydroxylation of progesterone and 17α-hydroxyprogesterone, cytochrome P45O, pH maxima and NADPH-cytochrome c reductase activity. Results of the study demonstrate that separation of tissues can be achieved by scraping the loosely adherent cortical cells away from the cells which adhere tightly to the capsule with a sharp scalpel. Microscopic examination revealed homogeneous glomerulosa and fasciculata cell populations. Microsomes prepared from cell populations separated in this way revealed the following: glomerulosa cell microsomes had virtually no C21 steroid 17α-hydroxylase activity while fasciculata cell micromes were active in both 17α- and 21-hydroxylation; the maximum activity in the pH range tested for 21-hydroxylation was found to occur between 6.5-7.5 with no difference between fasciculata and glomerulosa; with equal concentrations of antiserum to NADPH-cytochrome c reductase, reaction rates of 21-hydroxylation were inhibited by 65% and NADPH-cytochrome c reductase by 40% in both glomerulosa and fasciculata microsomes. This study demonstrates a method by which C21-steroid 21-hydroxylation may be analyzed without significant interference from 17α-hydroxylation reactions.
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