Labeling of Cysteine 231 in Acetylcholinesterase from Torpedo nobiliana by theActive-Site Directed Reagent, 1-Bromo-2-(14C) pinacolone

摘要

Acetylcholinesterase (AcChE, EC 3.1.1.7) was isolated from the electric organ ofT. nobiliana and treated with the active-site-directed alkylating agent 1-bromo-2-(14C)pinacolone or with BrPin, which acts initially as a competitive inhibitor, and then inactivates the enzyme. AcChE aliquots were digested with trypsin and fractionated by reversed phase high performance liquid chromatography. Inactivation caused a decrease in one absorption peak and an increase in another, identified as the peptide beginning at Ala-222 and extending to Arg-242. 5-Trimethylammonio-2-pentanone, a competitive inhibitor, isosteric with acetylcholine, retarded the inactivation and decreased the quantity of labeled peptide.