Genetic Aspects of Cortisol Hydroxylation in Guinea Pigs: Kinetics of Enzymatic 2α- and 6β-Hydroxylation by Adrenal Homogenates11

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Abstract
Hydroxylation of 4-14C-cortisol at C-6[beta] by adrenal and liver homogenates from 2 strains of guinea pigs (high and low producers), distinguished by high and low urinary excretion of hydroxylated cortisol derivatives, has been demonstrated. The radiochemical homogeneity of the 6[beta]-hydroxycortisol formed was established by chromatographic separations and degradation to 11[beta]-hydroxyandrqst-4-ene-3, 17-dione. Methods have been presented to study the kinetics of enzymatic 2-[alpha] and 6[beta]-hydroxylation of 4-14c-cortisol by guinea pig adrenal homogenates. With both high and low producers at 10[mu]M substrate concentration, the optimal pH of 2[alpha]-and 6[beta]-hydroxylation was in the range of 7. 6-8. 0 and maximal hydroxy-lation occurred at a TPNH concentration of 0. 50-0. 65 mM. Under these optimal conditions, linear progress curves of 2[alpha]- and 6[beta]-hydroxylation have been been found with adrenal homogenates from high producers at tissue concentrations of 10-40 mg/ml and incubation times up to 15-20 min. allowing calculation of initial rates from either the slopes or single points, and the rates were proportional to tissue concentration. At tissue concentrations of 50 mg/ml and higher the metabolism of substrate exceeded 80% at 15 min. leading at concentrations higher than 50 mg/ml to nonlinear 2[alpha]-hydroxylation progress curves (with maxima) and to rates no proportional to tissue concentration. The low producers exhibited linear progress curves of 2[alpha]-hydroxylation at tissue concentrations of 10-50 mg/ml up to 16 min. of incubation, whereas the progress curves of 6[beta]-hydroxylation were linear only at 10 and 20 mg/ml. The initial rates of 2[alpha]- and 6[beta]-hydroxylation by the low producers exhibited considerable devia-tions from linearity with respect to tissue concentration above 20 mg/ml. Adrenal homogenates from high producers exhibited a biomodal 1/V vs. 1/S plot indicating 2 apparent Km constants for 2[alpha]-hydroxyla-tion of 0. 33 mM and 9. 2 [mu]M, while the low producers revealed a single Km of 10. 6 [mu]M. Comparison of initial enzymatic rates by adrenal homogenates from high and low producers at tissue concentrations of 10-20 mg/ml revealed a 9. 2- and a 6. 6-fold higher 2[alpha]- and 6[beta]. hydroxy-lation activity, respectively, by the high producers.