Glycerol-3-phospho-D-myo-inositol 4-phosphate (Gro-PIP) is an inhibitor of phosphoinositide-specific phospholipase C.
The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.
Cruz-Rivera
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M
Department of Pharmacology, SmithKline Beecham, King of Prussia 19406.
Bennett
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Crooke
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D000215:Acylation; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005994:Glycerophosphates / Q000494:pharmacology*; D006168:Guinea Pigs; D006868:Hydrolysis; D007295:Inositol Phosphates; D007700:Kinetics; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D014599:Uterus / Q000201:enzymology / Q000378:metabolism*
D005994:Glycerophosphates; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D010726:Phosphodiesterase Inhibitors; C062528:glycero-3-phosphoinositol 4-phosphate; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase