Two‐Step Immunoaffinity Purification of Acetylcholinesterase from Rabbit Brain

K. P. Mintz, S. Brimijoin

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X‐100 from rabbit brain was purified 2,000‐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 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver‐staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogenous AChE to be completed in fewer than 7 days, at yields exceeding 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 molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (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 two‐step immunopurification procedure presented in this communication offers a convenient route to homogenous neural AChE in quantities useful for detailed biochemical and immunochemical study.

Original languageEnglish (US)
Pages (from-to)225-232
Number of pages8
JournalJournal of neurochemistry
Volume44
Issue number1
DOIs
StatePublished - Jan 1985

Keywords

  • Acetylcholinesterase
  • Immunoaffinity chromatography
  • Monoclonal antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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