GTP-dependent hydrolysis of phosphatidylinositol-4,5-bisphosphate by a soluble phospholipase C from adult human epidermis

The effects of tumor promoting phorbol esters, which activate protein kinase (C (PK-C), on epidermis suggest that PK-C is important in the regulation of epidermal growth and differentiation. Since in vivo PK-C is activated by the products of phospholipase C (PL-C)-catalyzed hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), they have investigated the properties of this reaction. Soluble PL-C from adult human epidermis was incubated with sonicated lipid vesicles containing (3H-inositol)PIP2, for 10 minutes at 370C. Water soluble reaction products were extracted, chromatographed and quantitated. In the presence of physiological concentrations of Ca++ magnesium and GTP PIP2, but not phosphatidylinositol, was hydrolyzed by PL-C (11.7 nmol/min/mg). Addition of GTP or GTPγS stimulated activity greater than 15 fold. Half maximal and maximal activity were observed at 10 μM and 100 μM GTPγS, respectively. ATP was unable to substitute for GTP, and GDP/S inhibited PIP2 hydrolysis in a dose dependent manner. Activity was sensitive to pH, and exhibited a sharp optimum at pH 6.5. In addition, the PL-C preparation specifically bound (35S)GTP S. These data demonstrate that adult human epidermis contains PL-C activity that specifically hydrolyzes PIP2 and suggest the involvement of a GTP-binding regulatory protein in this reaction