Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11β-/18-hydroxylase from porcine adrenal

Abstract

Highly purified cytochrome P-450 11β-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [ 3H] aldosterone from [ 3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11β-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11β-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H + and 3 moles of oxygen per mole of aldosterone produced: C 21 H 30 O 3 + 3 NADPH + 3 H + + 3 O 2 → C 21 H 28 O 5 + 3 NADP + + 4 H 2 O