Structural requirements of phosphatidylinositol-specific phospholipase C delta 1 for enzyme activity.

Abstract

Phosphatidylinositol-specific phospholipase C delta 1 isozyme of phosphoinositol-specific phospholipase C has been used for studies of structural requirements for the catalytic function. The enzyme was expressed in a bacterial system and purified to homogeneity. Using a combination of deletion mutant analysis and limited proteolysis, it was found that the large proportion of the molecule participated in formation of a catalytic domain (residues 139-756); it included regions of high and low conservation with other phospholipase-C molecules. These studies also showed that the residues spanning regions of conservation, designated as X and Y, were exposed and highly susceptible to proteolysis by trypsin. Two of the fragments resulting from the cleavage (30 kDa and 40 kDa) interacted and, under non-denaturing conditions, formed a protein of 70 kDa.