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

摘要

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 6#beta#- and 16#alpha#-hydroxylation) in koala liver were similar to or reduced relative to rat. Hepatic microsomal NADPH-supported 17#beta#-hydroxysteroid oxidoreductase (17#beta#-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 17#beta#-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 #mu#M (n = 4) irrespective of the cofactor used, whilst the K_m for NADPh was 0.04-0.05 #mu#M (n = 2) and for NADH was 134-430 #mu#M (n = 2). Potential inhibitors were evaluated for their effects on NADPH-mediated 17#beta#-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 #mu#M).