title: Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.
abstract: Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.
authors.affiliation: Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: M T
authors.initials: MT
authors.lastname: Lehto
authors.forename: F J
authors.initials: FJ
authors.lastname: Sharom
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D001413:Bacillus thuringiensis / Q000201:enzymology*; D002462:Cell Membrane / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism*; D008565:Membrane Proteins; D010743:Phospholipids / Q000494:pharmacology; D013552:Swine; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008081:Liposomes; D008565:Membrane Proteins; D010743:Phospholipids; D000249:Adenosine Monophosphate; D015720:5'-Nucleotidase; D010738:Type C Phospholipases