Cholinesterases from flounder muscle. Purification and characterization of glycosyl-phosphatidylinositol-anchored and collagen-tailed forms differing in substrate specificity.
Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen-tailed asymmetric acetylcholinesterase sedimenting at 17S, 13S and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6S in the presence of Triton X-100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross-reacting determinant, which is found in the hydrophilic form of a number of glycosyl-phosphatidylinositol-anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The Km and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The p(IC50) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent-soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 x 10(7) h-1, and 1 active site/subunit were determined.
Stieger
S
S
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Gentinetta
R
R
Brodbeck
U
U
D000215:Acylation; D000818:Animals; D001588:Benzoylcholine / Q000031:analogs & derivatives; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D002499:Centrifugation, Density Gradient; D002802:Cholinesterases / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism*; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D005399:Fishes; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011092:Polyethylene Glycols; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D013860:Thiocholine / Q000031:analogs & derivatives; D010738:Type C Phospholipases
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; C518182:benzoylthiocholine; D001588:Benzoylcholine; D011092:Polyethylene Glycols; D013860:Thiocholine; D003094:Collagen; D002802:Cholinesterases; D010738:Type C Phospholipases; D012697:Serine Endopeptidases; D019286:Endopeptidase K