Isolation and Purification of a 5α-Hydroxysterol Dehydrase of Yeast

Abstract

Abstract An enzyme that catalyzes the anaerobic dehydration of ergosta-7,22-diene-3β,5α-diol to ergosterol has been isolated from baker's yeast (Saccharomyces cerevisiae). The enzyme has been purified about 500-fold by ammonium sulfate precipitation, chromatography on Sephadex G-100, and adsorption onto calcium phosphate gel. No cofactors are required for activity; 2-mercaptoethanol was added to all buffers. Throughout purification, phospholipid and ergosterol are associated with the active enzymic fraction. The lipid may be removed by digestion with phospholipase A; gross changes in the physical appearance of the purified enzyme-phospholipid complex are observed following phospholipid digestion. However, the phospholipid may be completely digested without significant loss of dehydrase activity. Many assays of the dehydrase activity have been developed. In two spectrophotometric assays, ergosterol formation is measured by absorbance at 282 nm, either directly in the reaction mixture or indirectly following saponification and extraction. Substrate disappearance and product formation have been measured both by quantitative gas-liquid chromatography and by conversion of 14C-labeled substrate into ergosterol. The rates of formation of ergosterol measured by each assay method are identical. The disappearance of substrate and the appearance of ergosterol are equimolar.