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

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 microM) 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-gamma-S) significantly stimulated hydrolysis of PIP2, but not PI. Stimulation of PIP2 hydrolysis by GTP was dose-dependent between 1-100 microM GTP. Other nucleoside triphosphates and nucleotide analogues were unable to substitute for GTP or GTP-gamma-S. A GTP-gamma-S-stimulated PIP2 hydrolysis was inhibited by guanosine-5'-O-(2-thiodiphosphate (GDP-beta-S). The phospholipase C preparation specifically bound [35S]GTP-gamma-S and this binding was also inhibited by GDP-beta-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).