Aspects of the determination of biphenyl hydroxylase activity in liver homogenates: II. Sex and species differences in the effect of mitochondria

Abstract

A study is presented of the influence of mitochondria on the hydroxylation of biphenyl by liver microsomes of male and female rats, dogs, and monkeys ( Macaca mulatta). Greater activity of biphenyl hydroxylase was seen in microsomes of male origin only in the case of monkey and dog, using NADP + and NADPH, respectively, as energy sources. According to species and sex of animal from which microsomes were derived, progressive increase in the standard concentration (0.1 m M) of NADP + present in the assay system to 0.5–5 m M elicited maximum stimulation of biphenyl hydroxylation ranging from 20 to 100%. Levels of 10 m M NADP + produced a slight decrease in enzyme activity, possibly due to substrate inhibition. Under comparable conditions, replacement of NADP + by up to 10 m M NADPH substantially increased the observed stimulation of biphenyl hydroxylation. Sex and species differences were again evident, and the greatest enhancement of activity seen was 20–140%. In general, the addition of various amounts of previously frozen mitochondria to the assay system led to proportional inhibition of biphenyl hydroxylation. Striking species and sex differences were observed in regard to microsomal susceptibility to the inhibitory effect of mitochondria, as measured also by the capacity of progressively increasing levels of NADP + or NADPH, added to the assay system, to overcome the inhibition produced by different amounts of mitochondria. Judged by these criteria, microsomes from the dog were least susceptible, and those from the female rat were most vulnerable, to mitochondrial inhibition. In both rat and monkey there was a distinct sex difference in microsomal response, such that with microsomes from male monkeys the addition of mitochondria in the presence of 0.05 m M NADP + actually stimulated biphenyl hydroxylation. In relation to the sex and species differences in microsomal susceptibility, the observed sex difference in mitochondrial inhibitory activity is noteworthy, as also are the differences between the effectiveness of NADP + and NADPH in overcoming such inhibition.