Studies on the biosynthesis of 18-oxygenated steroids from exogenous corticosterone by domestic duck ( Anas platyrhynchos) adrenal gland mitochondria

The transformation of exogenous, isotopically labelled corticosterone to 18-hydroxycorti-costerone and aldosterone by domestic duck ( Anas platyrhynchos) adrenal gland mitochondria was studied. The mitochondrial 18-oxygenating system was NADPH dependent. K +, Na +, Ca 2+ and Mg 2+ were necessary for maximal enzymatic activity. The enzyme present in 1 mg of mitochondrial protein became saturated with 13.2 βM of substrate (13.8 μg). Under saturation conditions the maximal production of 18-hydroxycorticosterone was found as 1.43 nmole/min/ mg protein and that of aldosterone 0.33 nmole/min/mg protein. The Michaelis constant for both 18-hydroxycorticosterone and aldosterone was 6.6 × 10 −6M. Q 10 (average between 20°C and 40°C) was 2.16 for the reaction corticosterone → 18-hydroxycorticosterone and 1.34 for the reaction corticosterone → aldosterone. The mitochondrial enzyme system did not 18-oxygenate exogenous 11-deoxycorticosterone, 11-dehydrocorticosterone, 20β-dihydro-corticosterone. Exogenous 18-hydroxycorticosterone and aldosterone were not metabolized. The kinetics of the transformation of corticosterone to 18-oxygenated metabolites suggested a parallel pseudo-first order reaction rather than a series pseudo-first order reaction. 18-Oxygenation of corticosterone was strongly inhibited by d,1-18-hydroxycorticosterone, p-chloromercuribenzoate, carbon monoxide, metopirone (competitive inhibition; K i = 3.0 × 10 −6 M for 18-hydroxycorticosterone and 9.0 × 10 −6M for aldosterone) and by aminopterin (noncompetitive inhibition; K i = for both metabolites 2.0 × 10 −5M). Protein synthesis inhibitors did not have any effect. Cytochrome-P450 was shown to be present in mitochondria by spectrophotometric measurements. Addition of corticosterone. 11-deoxycorticosterone and metopirone produced type II difference spectra. The mitochondrial P450 became saturated with either corticosterone or metopirone at a concentration of 21.8 nmoles/mg protein. From these studies it was concluded that the duck adrenal mitochondrial 18-oxygenating system differed from the mammalian adrenal system previously described, especially in regard of substrate specificity. 18-Hydroxycorticosterone, either endogenous or exogenous, could not serve as substrate for aldosterone production with duck adrenal mitochondria. However, under ordinary circumstances. 18-hydroxycorticosterone synthesis was always associated with aldosterone synthesis in rather fixed proportions. Circumstantial evidence sugge