Purification and Quantification of Saccharomyces cerevisiae Cytochrome P450
Cytochrome P450s (cyt.P450s) are a class of hemoprotein enzymes found in a wide variety of organisms, where they play an important role in endogenous metabolism and the metabolism of xenobiotic compounds. In eukaryotes these enzymes are membrane-bound, in most cases located in the endoplasmic reticulum. Higher eukaryotes often have complex systems containing many cyt.P450s. However, vegetatively growing Saccharomyces cerevisiae contains only one major cyt.P450, the lanosterol 14α demethylase (1), and thus provides an ideal model system for the study of microsomal eukaryote cyt.P450s. Methods will be described for the quantification of yeast cyt.P450s, and the purification of the main yeast cyt.P450. This latter method was first described by Yoshida and Aoyama (1), and King et al. (2). Recently, the mRNA of a second S. cerevisiae cyt.P450 gene has been detected among sporulation-specific transcripts (3).
KeywordsAmmonium Sulfate Microsomal Protein Sodium Dithionite Sodium Cholate Microsome Preparation