Activation of polyphosphoinositide phospholipase C by guanosine 5′- O-(3-thio)triphosphate and fluoroaluminate in membranes prepared from a human T cell leukemia line, JURKAT

Abstract

Polyphosphoinositide hydrolysis was studied in membranes prepared from a human T cell leukemia line, JURKAT, prelabeled with myo-[2- 3H]inositol. The formation of inositol bis- and trisphosphates was stimulated in a buffer with 110 nM free Ca 2+ with a nonhydrolyzable GTP analogue, GTPγS, and NaF plus AlCl 3 in a time- and concentration-dependent manner. GTPγS and NaF-AlCl 3 had no significant effect on the inositol monophosphate level. AlCl 3 enhanced the NaF-stimulated release of inositol polyphosphates. Optimum concentrations of NaF and AlCl 3 produced 1.5-fold more inositol polyphosphates than that produced by optimum concentration of GTPγS. OKT3 monoclonal antibody, an antibody against the T-cell receptor complex, did not stimulate the inositol polyphosphate formation by JURKAT membranes even in the presence of GTP, although the antibody at the concentrations used markedly stimulated the hydrolysis of polyphosphoinositides in intact JURKAT cells.