Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes.
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.
Bolt
M J
MJ
Department of Medicine, University of Chicago, IL 60637.
Bissonnette
B M
BM
Wali
R K
RK
Hartmann
S C
SC
Brasitus
T A
TA
Sitrin
M D
MD
D000227:Adenine Nucleotides / Q000494:pharmacology; D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D001647:Bile Acids and Salts / Q000494:pharmacology; D015153:Blotting, Western; D002413:Cations, Divalent; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006150:Guanine Nucleotides / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D001647:Bile Acids and Salts; D002413:Cations, Divalent; D006150:Guanine Nucleotides; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D000408:Alamethicin; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase