Abstract
The intracellular concentration of cyclic AMP controls the biosynthesis of the microsomal cytochrome P-450 of Saccharomyces cerevisiae. The concentration of cyclic AMP in the cell is in turn related inversely to the extracellular concentration of glucose. When yeast was grown to the logarithmic phase in medium containing low concentrations of glucose and then transferred to high glucose medium rapid biosynthesis of cytochrome P-450 occurred, essentially under conditions of glucose repression of the mitochondrial cytochromes. The biosynthesis of cytochrome P-450 was studied in yeast protoplasts transferred to high glucose medium. This process v/as inhibited by actinomycin D and cycloheximide and therefore required de novo protein synthesis. Cylic AMP added to protoplasts suspended in the high glucose medium caused partial repression of cytochrome P-450 biosynthesis. Conversely, all previous reports of cyclic AMP effects in microorganisms have involved a positive effect on enzyme biosynthesis due to the lifting of glucose repression. The cytochrome P-450 produced by yeast was found to be capable of metabolising benzo(a)pyrene. The major metabolites, as identified by high-pressure liquid chromatography were 3-hydroxybenzo(a)pyrene, 9-hydroxybenzo(a)pyrene and (ii) 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene. Pretreatment of the yeast with benzo(a)pyrene during growth decreased the Michaelis constant (Km) of the aryl hydrocarbon hydroxylase for benzo(a)pyrene compared with control yeast, using either NADPH or cumene hydroperoxide as the cofactor. A solubilised and a solubilised and immobilised cytochrome P-450 preparation was capable of benzo(a)pyrene hydroxylation using cumene hydroperoxide as the cofactor. The interaction of benzo(a)pyrene with cytochrome P-450 was further investigated by means of an equilibrium gel filtration method using [G-3H] benzo(a)pyrene. There appeared to be twenty binding sites for benzo(a)pyrene in both yeast and rat liver microsomes. Benzo(a)pyrene was found to produce a modified type I spectral change with yeast and rat liver microsomes. Type II changes were obtained with imidazole and sodium phenobarbitone. Several drug-metabolising assays of cytochrome P-450 activity were investigated but none proved to be satisfactory.