Purification, Properties, and Phosphorylation by Protein Kinase C of Two Phosphoinositidase C Isozymes from Rat Brain

: Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel elec-trophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-β-1 and PLC-γ of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-β-1 and PLC-γ on Mono Q. Purified rat brain protein kinase C phosphorylated PLO-γ but not PLC-β-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphory-lation of another protein in the preparations.