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 phosphoinositidespecific 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-α-Glycerophospho-d-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-p), l-α-glycerophospho-d-myo-inositol (Gro-PI), or l-α-glycerophospho-d-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 μM [3H]phosphatidylinositol ([3H]PI), 10 μM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 μM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP]revealed an IC50 = 380 μM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 μM and 300 μM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 μM. Ca2+ concentrations over the range 1.1 μM 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 μM and 500 μM [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 μM [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. © 1990.