Abstract
The effect of glucose concentration in growth medium on biosynthesis of cytochrome P-450/P-448 under aerobic and anaerobic conditions was examined. High glucose concentration was required in both cases. The cytochrome P-450 enzyme from yeast Saccharomyces cerevisiae showed a Soret peak in the reduced CO-difference spectrum at 448nm, was purified to homogenity and had a molecular weight of 55,500. Cytochromes c(P-450) reductase, b[5], b[5] reductase were also purified from the same microsomal preparation. Amino acid analysis of yeast cytochrome P-448 revealed the presence of 407 residues per molecule. The sum of molecular weight of the polypeptides and other components was in good agreement with the molecular weight value obtained from SDS-polyacrylamide gel electrophoresis. A reconstituted system of purified yeast cytochrome P-448, NADPH:cytochrome c(P-450) reductase and phospholipid showed benzo(a)pyrene hydroxylase activity. The spin state of substrate-free and benzo(a)pyrene-bound purified yeast cytochrome P-448 were 94% and 82% low spin at 22°C respectively. Equilibrium gel filtration analysis of the number of benzo(a)pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. In addition to benzo(a)pyrene, lanosterol, ethylmorphine, dimethylnitrosamine and sodium phenobarbitone showed Type I binding spectra with yeast cytochrome P-448. A more specific and efficient form of benzo(a)pyrene hydroxylase was induced by the addition of benzo(a)pyrene to the yeast growth medium at zero time. Based on the effect of modification of various amino acids on parameters of yeast cytochrome P-448, participation of -SH group(s) and tyrosyl residue(s) in the active site of this enzyme is suggested. Various supports were evaluated for immobilization of yeast cytochrome P-448. Calcium alginate was found to be especially useful. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the cytochrome P-450 family. This enzyme, however, has many properties in common with cytochrome P-448 from mammalian sources.