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 (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] 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, 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. approximately 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.
Camps
M
M
Pharmakologisches Institut, Universität Heidelberg, Federal Republic of Germany.
Hou
C F
CF
Jakobs
K H
KH
Gierschik
P
P
D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D003840:Deoxycholic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D006098:Granulocytes / Q000201:enzymology / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007295:Inositol Phosphates / Q000096:biosynthesis / Q000097:blood; D007700:Kinetics; D007942:Leukemia, Experimental / Q000097:blood* / Q000473:pathology; D007951:Leukemia, Myeloid / Q000097:blood* / Q000473:pathology; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000096:biosynthesis / Q000097:blood*; D010743:Phospholipids / Q000097:blood; D013268:Stimulation, Chemical; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000097:blood
D007295:Inositol Phosphates; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010743:Phospholipids; D003840:Deoxycholic Acid; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases