Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes

• Published in: Biochemical journal Vol. 292; no. 1; pp. 271 - 276
• Main Authors: BOLT, M. J. G, BISSONNETTE, B. M, WALI, R. K, HARTMANN, S. C, BRASITUS, T. A, SITRIN, M. D
• Format: Journal Article
• Language: English
• Published: Colchester Portland Press 15-05-1993
• Subjects: Adenine Nucleotides - pharmacology; Alamethicin - pharmacology; Animals; Bile Acids and Salts - pharmacology; Biological and medical sciences; Blotting, Western; Cations, Divalent; Colon - cytology; Colon - drug effects; Colon - enzymology; Fundamental and applied biological sciences. Psychology; Guanine Nucleotides - pharmacology; Intestine. Mesentery; Intracellular Membranes - drug effects; Intracellular Membranes - enzymology; Isoenzymes - metabolism; Male; Microsomes - drug effects; Microsomes - enzymology; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Diacylglycerol-Lyase; Phosphatidylinositols - metabolism; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases - metabolism; Rats; Rats, Sprague-Dawley; Substrate Specificity; Vertebrates: digestive system; Rat; Digestive system; Enzyme; Isozyme; Rodentia; Microsome; Phospholipase C; Vertebrata; Brush border; Mammalia; Enzymatic activity; Animal; Colon; Laterobasal membrane
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2920271

The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5'-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5'-[beta-thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca2+ was biphasic, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca2+ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol.