Effects of heparin and other chemicals on phosphorylation of phosphatidylinositol in pig erythrocyte membrane

In the presence of Mg2+, the pig erythrocyte membranes were incubated with [gamma-32P]ATP at 30 degrees C for 3 min to study the effects of some chemicals on the phosphorylation of phosphatidylinositol. The incubations were stopped by the addition of chloroform/methanol (2:1, vol/vol) and the phospholipids were extracted with acid chloroform/methanol and separated on silica gel TLC plates. [gamma-32P] phosphatidyl-inositol-4-phosphate was quantitated by scintillation counting and autoradiography. The results indicated that heparin and neomycin inhibited the phosphorylation of phosphatidylinositol in a concentration-dependent manner, while dimethylsulfoxide stimulated the phosphorylation at low concentration (less than 10%), but inhibited at high concentration (greater than 10%). Hexachlorocyclohexane stimulated the phosphorylation within a certain limit of concentration up to 6.4 micrograms/ml. Li2SO4 (10 mmol/L), EGTA (100 mumol/L) and theophylline (100 mumol/L) had no significant effects.