Hepatic microsomal enzyme activity in the koala and tammar wallaby: high 17β -hydroxysteroid oxidoreductase activity in koala liver microsomes
I. Stupans ^a,*, S. Kong ^a, A. Kirlich ^a, M. Murray ^b, E.L. Bailey ^a, B.R. Jones ^a, R.A. McKinnon ^a
a) Center for Pharmaceutical Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA
b) School of Physiology and Pharmacology, University of New South Wales, Kensington, NSW
We have studied the hepatic microsomal xenobiotic metabolising capacity of koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii). Total cytochrome P450 content in hepatic microsomes from koala (0.87 ± 0.18 nmol/mg protein, n=4, mean (S.D.) and rat were comparable while tammar wallaby displayed reduced P450 content (0.24 ± 0.04 nmol/mg protein). Associated microsomal activities (NADPH cytochrome P450 reductase, aminopyrine N-demethylation, aniline hydroxylation, and androstenedione 6β- and 16α-hydroxylation) in koala liver were similar to or reduced relative to rat. Hepatic microsomal NADPH-supported 17β-hydroxysteroid oxidoreductase (17β-HSOR) activity was signiﬁcantly higher in koala (9.99 ± 3.08 nmol/mg protein/min) than in tammar wallaby liver (0.86 ± 0.16 nmol/mg protein/min). However, when NADH was utilised as cofactor the activity was similar in both marsupial species (koala, 1.44 ± 0.84 nmol/mg protein/min; tammar wallaby, 1.52 ± 0.44 nmol/mg protein/min). Michaelis–Menten parameters for the kinetics of 17β-HSOR androstenedione reduction by NADPH and NADH were determined in the koala. The Km for androstenedione was of the order of 1.9–4 µM (n=4) irrespective of the cofactor used, whilst the Km for NADPH was 0.04–0.05 µM (n=2) and for NADH was 134–430 µM (n=2). Potential inhibitors were evaluated for their effects on NADPH-mediated 17β -HSOR activity with menadione and, to lesser extents, menthone, benzaldehyde and metyrapone eliciting signiﬁcant inhibition. From detailed kinetic studies menthone was found to be an uncompetitive inhibitor of the activity in koala liver (Ki 220 µM).