Mechanisms responsible for the thermal sensitivity of adrenal microsomal monooxygenases

Studies were done to determine the mechanism(s) responsible for the thermal lability of adrenal microsomal monooxygenases. Preincubation of guinea pig adrenal microsomal suspensions at 37 degrees C caused large time-dependent declines in benzo(a)pyrene (BP) hydroxylase and benzphetamine (BZ) demethylase activities. Similar preincubations with hepatic microsomes had little effect on enzyme activities. The decreases in adrenal enzyme activities were completely prevented by co-incubation of microsomes with cytosol, but were not diminished by reduced glutathione, ascorbic acid, or bovine serum albumin. Partial protection was afforded by EDTA, suggesting that lipid peroxidation might be involved, but malonaldehyde production was not demonstrable and MnCl2, a potent inhibitor of lipid peroxidation, did not affect the decline in enzyme activities. The decreases in the rates of BP and BZ metabolism were prevented by including NADPH or NADP+ in the preincubation medium. The preincubation conditions causing losses of adrenal enzyme activities did not affect cytochrome P-450 concentrations or substrate binding to cytochromes P-450, as indicated by type I difference spectra. NADH-cytochrome c reductase activity also was not affected, but there were decreases in NADPH-cytochrome c reductase activity that were proportionately similar to the declines in drug-metabolizing activities. Direct assessment of NADPH-cytochrome P-450 reductase revealed similarly large decreases in enzyme activity resulting from preincubation of adrenal microsomes. The results demonstrate a need for extra caution when doing preincubation experiments with adrenal microsomal preparations, and suggest that the thermal lability of adrenal monooxygenases is attributable to effects at the active site of NADPH-cytochrome P-450 reductase.