Acetylcholinesterase of Schistosoma mansoni: purification and characterization.
Larval acetylcholinesterase (acetylcholine acetylhydrolase) EC 3.1.3.7 of the trematode Schistosoma mansoni was characterized and purified by affinity chromatography. The enzyme was solubilized from sonicated cercarial tissue and showed a Km value of 1.83 mM and a Vmax value of 102 U/mg protein. It was characterized as a true AChE since it hydrolyses acetylthiocholine more than seven times faster than butyrylthiocholine, and since it was inhibited by high concentrations of substrate. The enzyme was purified by affinity chromatography on a Sepharose column of the inhibitor [N-(6-aminocaproyl-6-aminocaproyl)-m-aminophenyl] trimethyl ammonium. The purified enzyme eluted from the column by decamethonium bromide migrated as a single band of 500 kD on nondenaturing polyacrylamide gel electrophoresis (PAGE), whether stained for proteins or for enzymatic activity. Analysis by SDS-PAGE revealed two major polypeptide bands of 76 kD and 30 kD. By labeling the enzyme with 3H-DFP (di-isopropyl-fluorophosphate), the 30-kD polypeptide was shown to contain the active site of the enzyme, with an additional labeled band of 110 kD also being detected. On the basis of our data we suggest that the principal species of S. mansoni AChE is a tetramer of four subunit polypeptides each of MW ca. 110 kD which are not linked by disulfide bonds, and which are further cleaved into two fragments, one of MW 76,000 and one of MW 30,000, the latter bears the active site.
D000110:Acetylcholinesterase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D051379:Mice; D008813:Mice, Inbred ICR; D008970:Molecular Weight; D012550:Schistosoma mansoni / Q000201:enzymology*; D012908:Snails; D013379:Substrate Specificity
D002800:Cholinesterase Inhibitors; D000110:Acetylcholinesterase