Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase.
Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.
Nichol
C P
CP
Laboratory of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Roufogalis
B D
BD
D000110:Acetylcholinesterase / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000378:metabolism; D050356:Lipid Metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011092:Polyethylene Glycols / Q000378:metabolism; D012995:Solubility; D010738:Type C Phospholipases / Q000378:metabolism
D010716:Phosphatidylinositols; D003208:Concanavalin A; D011092:Polyethylene Glycols; D000110:Acetylcholinesterase; D010738:Type C Phospholipases