GUANOSINE 5'-[GAMMA-THIO]TRIPHOSPHATE-STIMULATED HYDROLYSIS OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE IN HL-60 GRANULOCYTES - EVIDENCE THAT THE GUANINE-NUCLEOTIDE ACTS BY RELIEVING PHOSPHOLIPASE-C FROM AN INHIBITORY CONSTRAINT

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4,5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5'[γ-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 μM and 30 μM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5'-[β-thio]diphosphate, ATP, adenosine 5'-[γ-thio]triphosphate, UTP) did not affect phospholipase C activity. GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. ≃ 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.