Purification and characterization of an ascidian larval acerylcholinesterase.
Larval acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) of the ascidian Ciona intestinalis (L.) was purified by a two-step affinity chromatography procedure. Concavanalin A-Sepharose chromatography in batches provided the initial purification and was followed by chromatography on columns to which competitive inhibitors of acetylcholinesterase had been attached. The most efficient of these used m-carboxyphenylmethylammonium iodide coupled to Sepharose 4B via a hydrophobic 6-carbon spacer. In combination with the concanavalin A-Sepharose step, this affinity resin yielded recoveries of 30-39% with specific activities ranging from 580-730 units/mg protein, a total purification of 5000-7000-fold. Analysis of this product by polycrylamide gel electrophoresis in the presence of SDS and beta-mercaptoethanol revealed a single major polypeptide of M(r) 65 000-70 000. This protein was identified as the basic catalytic subunit of acetylcholinesterase by its coelectrophoresis with [(3)H]diisopropyl fluorophosphate-labeled enzyme. Sucrose density gradient studies demonstrated that the purified enzyme consisted of three distinct species that appeared to be qualitatively the same as those seen in crude extracts. The largest species (11 S) is possibly a tetramer of the basic catalytic subunit and the two smaller forms, the monomer and dimer. Purified enzyme was also used to produce anti-acetylcholinesterase antibody in rabbits. IgG prepared from the sera of immunized rabbits was shown to react completely (greater than 98%) with acetylcholinesterase from crude larval homogenates. This result also supports the conclusions that no qualitative selection occurred during the purification procedure and that the basic catalytic subunit is a fundamental component of all the larval acetylcholinesterases.
D000110:Acetylcholinesterase / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D002499:Centrifugation, Density Gradient; D002800:Cholinesterase Inhibitors; D002846:Chromatography, Affinity / Q000379:methods; D002938:Ciona intestinalis / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007814:Larva / Q000201:enzymology; D011817:Rabbits / Q000276:immunology; D014561:Urochordata / Q000201:enzymology*
D002800:Cholinesterase Inhibitors; D000110:Acetylcholinesterase