Two-Step Immunoaffinity Purification of Acetylcholinesterase from Rabbit Brain

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Abstract
Acetylcholinesterase (AChE; EC 3.1.1.17) extracted in 1% Triton X-100 from rabbit brain was purified 2000-fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium-Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2950 U/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only 1 silver-staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoresis homogenous AChE to be completed < 7 days, at yield > 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 showed little difference between the properties of the native and the purified enzyme. The MW of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protrein (Km = 0.16 .+-. 0.01 mM) were close to those of the native enzyme (Km - 0.12 .+-. 0.01 mM) when examined with acetylthiocholine iodide as substrate. The 2-step immunopurification procedure presented in this communication offers a convenient route to homogenous neural AChE in quantities useful for detailed biochemical and immunochemical study.