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

Abstract

The regulation of soluble phosphoinositide-specific phospholipase C from adult human epidermis by guanine nucleotide was investigated. In the presence of physiologic concentrations of Ca++ (1 μM) and Mg++ (1.5 mM), neither phosphatidylinositol (PI) nor phosphatidylinositol-4,5-bisphosphate (PIP2) were appreciably hydrolyzed. Addition of guanosine-5′-triphosphate (GTP) or guanosine-5′-O-(3-thiotriphosphate) (GTP-γ-S) significantly stimulated hydrolysis of PIP2, but not PI. Stimulation of PIP2 hydrolysis by GTP was dose-dependent betwen 1 - 100,μM GTP. Other nucleoside triphosphates and nucleotide analogues were unable to substitute for GTP or GTP-γ-S. A GTP-γ-S-stimulated PIP2 hydrolysis was inhibited by guanosine-5′-O-(2-thiodiphosphate (GDP-β-S). The phospholipase C preparation specifically bound [35s]GTP-γ-S and this binding was also inhibited by GDP-β-S. In addition to a 41,000-dalton pertussis toxin substrate, the phospholipase C preparation contained 3-4 GTP binding proteins with molecular weights between 20,000-30,000.These data demonstrate that human epidermis contains a soluble GTP-dependent phospholipase C activity that specifically hydrolyzes PIP2 and suggest that this reaction is regulated by a GTP-binding protein(s). © 1989.