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 purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-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 these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected 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)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.
James
S R
SR
Department of Biochemistry, University of Dundee, U.K.
Demel
R A
RA
Downes
C P
CP
D000818:Animals; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D014422:Turkeys; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D018129:Phosphatidylinositol Phosphates; D010761:Phosphorus Radioisotopes; C037178:phosphatidylinositol 4-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C068961:phosphatidylinositol 4,5-biphosphate kinase; D010738:Type C Phospholipases