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.