Fast and efficient purification of yeast plasma membranes using cationic silica microbeads

Abstract

A fast and efficient procedure for the purification of plasma membranes of Saccharomyces cerevisiae is described. Protoplasts served as starting material. They were coated with cationic silica microbeads. After lysis, the plasma membranes were washed free from debris and cell organelles. This procedure resulted in a high yield (about 85%) of plasma membranes, as judged by measuring vanadate-sensitive ATPase as a plasma membrane marker. The enzyme was enriched 12-fold relative to the homogenate after lysis. Its specific activity was 1.5--2.0 micromol/min per mg protein, the pH optimum was 6.5, and 10 microM vanadate was sufficient to obtain maximum inhibition. Based on the assay of internal markers and electron microscopic studies, we found our preparation essentially free of contamination from other cell organelles.