Hepatic microsomal enzyme activity in the koala and tammar wallaby: high 17β-hydroxysteroid oxidoreductase activity in koala liver microsomes

Abstract

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 K m for androstenedione was of the order of 1.9–4 μM ( n=4) irrespective of the cofactor used, whilst the K m 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 ( K i 220 μM).