Purification of phosphoinositide‐specific phospholipase C from a particulate fraction of bovine brain

Abstract

The coupling of various agonist receptors to the hydrolysis of phosphoinositides has generated much interest in the nature of the phospholipase C that is activated. Here we report the purification of a bovine brain phospholipase C derived from the particulate fraction. A 1000-fold purification was achieved by a combination of heparin-Sepharose, DEAE-cellulose and gel-permeation chromatography. The purified enzyme appears to be monomeric and under denaturing conditions shows a single staining major polypeptide of molecular mass 154 kDa in SDS gels. The enzyme is specific for phosphoinositides although it shows a marked preference for the polyphosphoinositides. With phosphatidylinositol 4,5-bisphosphate as substrate the enzyme expresses a specific activity of > 100 μmol min⁻¹ mg⁻¹. The phospholipase C is activated by Ca²⁺ (0.1 – 10 μM). The behaviour of this particulate enzyme is discussed in the context of agonist-induced phosphatidylinositol hydrolysis.