Phosphoinositide hydrolysis by guanosine 5'-[gamma-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes

Abstract

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)