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'-gamma-thio]triphosphate (GTPS]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTPS] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5'-beta-thio]diphosphate. ATP, adenosine 5'-gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTPS] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTPS] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTPS]-stimulated phospholipase C activity. In the absence of GTPS], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTPS] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTPS]-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) GTPS] relieves this inhibition, most likely by activating a soluble GTP-binding protein.