INTERFACIAL HYDROLYSIS OF PHOSPHATIDYLINOSITOL 4-PHOSPHATE AND PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE BY TURKEY ERYTHROCYTE PHOSPHOLIPASE-C

The activity of a beta-isoform of phospholipase C (PLC) partially puri fied from turkey erythrocyte cytosol was assayed using phospholipid mo nolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and hepari n chromatographies. P-33-labelled substrates were prepared using parti ally purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using suc h P-33-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under the se conditions, PtdIns4P hydrolysis obeyed approximately first-order ki netics whereas PtdIns(4,5)P-2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly aff ected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P- 2 the maximum activity occurred at the markedly higher surface pressur e of 30 mN/m. These data are discussed in terms of the substrate speci ficity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.