O-Aacetyl-l-serine-O-acetyl-l-homoserine sulfhydrylase from Saccharomyces cerevisiae

Abstract

The physiological function of O-acetylserine-O-acetylhomoserine sulfhydrylase of Saccharomyces cerevisiae is the biosynthesis of both cysteine and homocysteine. The products of these reactions, cysteine and homocysteine, are determined after removing sulfide at an acidic pH by reaction with nitroprusside. Other spectrophotometric determinations of cysteine and homocysteine are also convenient. Use of radioactive sulfide ( 35 S) is recommended for an accurate determination of the activity in crude extracts. In the course of extraction by autolysis, Pressed yeast cake, 5 kg, is crumbled, spread to a thickness of 2-3 cm over stainless steel trays, and mixed with an equal quantity of powdered Dry Ice. Thus frozen the cells are thawed at 30° with the aid of an electric fan. The freezing-thawing procedure is repeated once. The resulting thick cell suspension is dried at room temperature in front of an electric fan and subsequently kept under reduced pressure over pellets of NaOH. The purification procedure has been found to be reproducible and yields one major protein band on disk gel electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate also yields a single band. Extraction of the enzyme can also be effectively carried out by agitating the cell suspension with glass beads in a Dyno Mill as described previously for extraction of low molecular weight O-ace-tylserine sulfhydrylase from a mutant-type strain of the same organism.