Purification and properties of a membrane-bound phospholipase B from baker's yeast (Saccharomyces cerevisiae).

Phospholipase B bound tightly to the membrane fraction of baker's yeast (Saccharomyces cerevisiae) was purified approximately 226-fold by extraction with sodium deoxycholate (DOC), acetone precipitation, ammonium sulfate fractionation, and column chromatographies on Phenyl-Sepharose CL-4B, DEAE-Sepharose CL-6B, and Sepharose 4B. Only one major activity peak with an apparent molecular weight of 330,000 was detected by gel filtration on Sepharose 4B. However, two glycoprotein bands, one major and one minor, were evident on SDS-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol. Isoelectric focusing also revealed two activities, pI 3.4 (major) and pI 3.0 (minor). The purified enzyme had phospholipase B activity (in the presence of DOC) and lysophospholipase activity in a ratio of 1:4.8. Both activities had an optimum pH of 3.5-4.0. Phospholipase B activity was appreciably stimulated only by DOC among bile acids, but lysophospholipase activity was markedly inhibited by them. Both activities were not stimulated by Ca2+ and inhibited by SDS, Triton X-100, Fe3+ and Al3+. The purified enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups. It preferentially hydrolyzed 1-acyl-sn-glycero-3-phosphocholine and, to a lesser degree, 2-acyl-sn-glycero-3-phosphocholine.