Purification of a phosphatidylinositol-glycan-specific phospholipase C from liver plasma membranes: a possible target of insulin action.
Insulin stimulates the hydrolysis of a phosphatidylinositol-glycan, resulting in the generation of two related inositol phosphate-glycan enzyme modulators and diacylglycerol. A phosphatidylinositol-glycan-specific phospholipase C that catalyzed this reaction was found in the plasma-membrane fraction of rat liver. The enzymatic activity was measured by the release of diacylglycerol from the glycosylated-phosphatidylinositol-membrane-anchored variant surface glycoproteins of African trypanosomes. The enzyme also catalyzed the production of an inositol phosphate-glycan from an insulin-sensitive phosphatidylinositol-glycan precursor. The enzyme was solubilized with neutral nonionic detergent and purified to near homogeneity by anion-exchange chromatography on DEAE-cellulose, followed by hydrophobic chromatography on butyl-agarose. The resulting enzyme preparation was purified approximately 20,000-fold from whole liver and exhibited one major silver-stained band of Mr 52,000 on NaDodSO4/PAGE. Gel permeation chromatography of the purified activity revealed a Stokes radius of 35 A and an apparent molecular weight of 62,000, suggesting that the enzyme was tightly associated with lipid or detergent but existed as a monomer in its active form. The enzyme was specific for glycosylated phosphatidylinositol; no hydrolysis of phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate, or phosphatidylcholine was observed. The enzyme was calcium independent and thiol activated. These data suggest a role for the phosphatidylinositol-glycan-specific phospholipase C as an effector for some of the metabolic actions of insulin.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D007328:Insulin; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase