A novel purification procedure for Penicillium notatum phospholipase B and evidence for a modification of phospholipase B activity by the action of an endogenous protease.

The purification of Penicillium notatum phospholipase B was greatly improved using phosphatidylserine-AH Sepharose 4B affinity chromatography. This chromatography simplified the procedure, stabilized the enzyme activity and gave six-fold higher yields and two-fold higher specific activity for the enzyme than the previous method (Kawasaki, N. and Saito, K. (1973) Biochim. Biophys. Acta 296, 426-430). During purification, it was found that an endogenous protease attacked phospholipase B and produced a modified type which exhibited extremely reduced phospholipase B (phosphatidylcholine hydrolysis) activity but maintained native lysophospholipase (lysophosphatidylcholine hydrolysis) activity. This protease was completely inhibited by phenylmethylsulfonylfluoride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that native and modified phospholipases B had exactly the same molecular weight (90 000) in the absence of beta-mercaptoethanol. However, the latter gave three bands in the presence of beta-mercaptoethanol, whereas the former only a single band. These results strongly suggested that the endogenous protease cleaved phospholipase B at only a few sites, thereby changing its phospholipase B activity.