CHARACTERIZATION OF PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE-C IN RAT COLONOCYTE MEMBRANES

The phosphoinositide signal transduction pathway mediates important pr ocesses in intestinal physiology, yet the key enzyme, phosphoinositide -specific phospholipase C (PI-PLC), is not well-characterized in the c olon. PI-PLC activity was examined in rat colonic membranes using exog enous [H-3]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or prod uct. The activity of membrane PI-PLC increased 6-fold with the additio n of alamethicin, and a further 2-3-fold enhancement was observed with 10 muM guanosine 5'-[gamma-thiol]riphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be spec ific, as up to 100 muM adenosine 5'-[gamma-thio]-triphosphate failed t o stimulate PI-PLC activity, and guanosine 5'-[beta-thio]diphosphate i nhibited activity. The response of membrane PI-PLC to Ca2+ was biphasi c, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparab le total PI-PLC activities and responses to GTP[S] and Ca2+ were obser ved in purified brush-border and basolateral membranes. Western immuno blots probed with monoclonal antibodies to PLC isoenzymes PLC-beta1, g amma1 and -delta1 demonstrated that these antipodal plasma membranes c ontain predominantly the PLC-delta1 isoform, with small amounts of PLC -gamma1 present but no detectable PLC-beta1. PLC-gamma1 was the major isoform detected in cytosol.