Abstract
Acetylcholinesterase (EC 3.1.1.7) from fetal bovine serum (FBS) was purified to electrophoretic homogeneity. The procedure involved procainamide affinity chromatography with native FBS, followed by chromatography on Sepharose 6B and DEAE-Sephadex. The acetylcholinesterase was purified approximately 44,000-fold, and 13 mg was obtained corresponding to an overall yield of about 45%. The purified acetylcholinesterase was stable at 4 degrees C for at least 8 weeks but was labile to freezing; however, in 50% glycerol the enzyme was stable at -20 degrees C for at least 12 weeks. FBS acetylcholinesterase exhibited typical substrate inhibition, had a Km of 120 microM, and a turnover number of 5300 s-1 with the substrate acetylthiocholine. The enzyme was highly sensitive to the specific acetylcholinesterase inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one. FBS acetylcholinesterase was characterized as a G4 form of acetylcholinesterase and was distinguished from bovine erythrocyte acetylcholinesterase on the basis of lectin gel binding, [3H] Triton X-100 binding, amino acid composition, number of catalytic subunits/molecule, and hydrodynamic properties. FBS acetylcholinesterase had a Stokes radius of 76 A as judged by gel filtration, and from this a molecular weight of 340,000 daltons was calculated. The enzyme had a subunit weight of approximately 83,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; paraoxon titration indicated a relative active site mass of 75,000 daltons. The amino acid composition of FBS acetylcholinesterase was similar to the human erythrocyte acetylcholinesterase (Rosenberry, T. L., and Scoggin, D. M. (1984) J. Biol. Chem. 259, 5643-5652). A monoclonal antibody directed against human erythrocyte acetylcholinesterase, AE-2, (Fambrough, D. M., Engel, A. G., and Rosenberry, T. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1078-1082) cross-reacted with FBS acetylcholinesterase.
Highlights
Acetylcholinesterase (EC 3.1.1.7) from fetal bovine gents [2,3]
Purification of Fetal Bovine Serum AcetylcholinesteraseThe first purification step consisted of adsorption of acetylcholinesterase from native forms which bind detergents show serum (FBS) to procainamide affinity gel
Selective elution withprocainamide resulted in the recovery of 90% of the applied acetylcholinesterase
Summary
Acetylcholinesterase (EC 3.1.1.7) from fetal bovine gents [2,3] Those globular forms which bind detergents show serum (FBS) was purified to electrophoretic homoge- a tendency to aggregate in the absence of detergents [3,4,5], neity. ’The abbreviations used are: RBC, erythrocyte; FBS, fetal bovine serum; ATC, acetylthiocholine; BW 284 C51, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one;SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DFP, diisopropyl fluorophosphate; mAb, monoclonal antibody. Immunological studies with monoclonal antibodies raised against human RBC acetylcholinesterase [23] suggested some homology between FBS acetylcholinesterase and othermammalian acetylcholinesterases
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