Abstract
An acetylcholinesterase inhibitor-Sepharose conjugate was prepared by coupling a derivative of the powerful acetylcholinesterase inhibitor, N-methylacridinium, to CNBr-activated Sepharose. Use of this conjugate permitted direct purification, by affinity chromatography, of the two molecular forms of acetylcholinesterase, 14 and 18 S, present in fresh electric organ tissue. The purified 14S and 18S acetylcholinesterases retained the capacity to aggregate at low ionic strength displayed by crude extracts of the enzyme. The major polypeptide components of the 14S and 18S enzymes, as revealed by acrylamide gel electrophoresis, closely resemble those observed in the 11S form of acetylcholinesterase, previously purified after tryptic digestion of electric-organ tissue.
Keywords: N-methylacridinium, protein subunits, proteolysis, acrylamide gel electrophoresis, sucrose gradients
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- Axén R., Porath J., Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature. 1967 Jun 24;214(5095):1302–1304. doi: 10.1038/2141302a0. [DOI] [PubMed] [Google Scholar]
- Changeux J. P. Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Mol Pharmacol. 1966 Sep;2(5):369–392. [PubMed] [Google Scholar]
- Cuatrecasas P., Wilchek M., Anfinsen C. B. Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci U S A. 1968 Oct;61(2):636–643. doi: 10.1073/pnas.61.2.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dudai Y., Silman I., Kalderon N., Blumberg S. Purification by affinity chromatography of acetylcholinesterase from electric organ tissue of the electric eel subsequent to tryptic treatment. Biochim Biophys Acta. 1972 Apr 7;268(1):138–157. doi: 10.1016/0005-2744(72)90208-2. [DOI] [PubMed] [Google Scholar]
- Dudai Y., Silman I. The subunits of an acetylcholinesterase preparation purified from trypsin-treated electric eel tissue. FEBS Lett. 1971 Sep 1;16(4):324–328. doi: 10.1016/0014-5793(71)80381-2. [DOI] [PubMed] [Google Scholar]
- KREMZNER L. T., WILSON I. B. A chromatographic procedure for the purification of acetylcholinesterase. J Biol Chem. 1963 May;238:1714–1717. [PubMed] [Google Scholar]
- Kalderon N., Silman I., Blumberg S., Dudai Y. A method for the purification of acetylcholinesterase by affinity chromatography. Biochim Biophys Acta. 1970 Jun 23;207(3):560–562. doi: 10.1016/s0005-2795(70)80020-4. [DOI] [PubMed] [Google Scholar]
- Leuzinger W., Baker A. L. Acetylcholinesterase, I. Large-scale purification, homogeneity, and amino Acid analysis. Proc Natl Acad Sci U S A. 1967 Feb;57(2):446–451. doi: 10.1073/pnas.57.2.446. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Massoulié J., Rieger F. L'acétylcholinestérase des organes électriques de poissons (torpille et gymnote); complexes membranaires. Eur J Biochem. 1969 Dec;11(3):441–455. doi: 10.1111/j.1432-1033.1969.tb00794.x. [DOI] [PubMed] [Google Scholar]
- Massoulié J., Rieger F., Tsuji S. Solubilisation de l'acetylcholinestérase des organes électriques de gymnote. Action de la trypsine. Eur J Biochem. 1970 Jul;14(3):430–439. doi: 10.1111/j.1432-1033.1970.tb00307.x. [DOI] [PubMed] [Google Scholar]
- Rosenberry T. L., Chang H. W., Chen Y. T. Purification of acetylcholinesterase by affinity chromatography and determination of active site stoichiometry. J Biol Chem. 1972 Mar 10;247(5):1555–1565. [PubMed] [Google Scholar]
- Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]