PURIFICATION OF A NOVEL PHOSPHOLIPASE-C ISOZYME FROM BOVINE CEREBELLUM

Abstract

An isozyme of phosphoinositide-specific phospholipase C (PLC) was purified to near homogeneity from bovine cerebellum by a combination of several column chromatography procedures. Approximately 80 mug of pure enzyme were obtained from 4 kg of bovine cerebellum, with a final specific activity of 7.5 mumol/min/mg protein in the presence of 0.1% deoxycholate. The enzyme is specific for phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but does not hydrolyze phosphatidylcholine. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis is approximately 97,000. Polyclonal antibodies to previously characterized PLC isozymes, PLC-beta1,beta2,gamma1,gamma2, and -delta1, did not cross-react with the purified cerebellar enzyme. Moreover, polyclonal antibodies prepared against the cerebellar enzyme did not react with purified PLC-beta1, -beta2, -gamma1, -gamma2, or -delta1. However, the cerebellar enzyme was recognized by two antibodies generated against peptide sequences common to mammalian PLC isozymes. Comparison of partial amino acid sequences of the purified cerebellar enzyme with the deduced amino acid sequence of each known PLC isozyme shows that the cerebellar enzyme is a novel PLC, which could be classified as a PLC-beta-type isozyme. Thus, we have designated this enzyme PLC-beta4.