Skip to main content
SpringerLink
Log in

Purification of Acetylcholinesterase by 9-Amino-1,2,3,4-tetrahydroacridine from Human Erythrocytes

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The acetylcholinesterase enzyme was purified from human erythrocyte membranes using a simple and effective method in a single step. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a well-known drug for the treatment of Alzheimer's disease, which inhibits cholinesterase. We have developed a tacrine ligand affinity resin that is easy to synthesize, inexpensive and selective for acetylcholinesterase. The affinity resin was synthesized by coupling tacrine as the ligand and l-tyrosine as the spacer arm to CNBr-activated Sepharose 4B. Acetylcholinesterase was purified with a yield of 23.5 %, a specific activity of 9.22 EU/mg proteins and 658-fold purification using the affinity resin in a single step. During purification, the enzyme activity was measured using acetylthiocholine iodide as a substrate and 5,5′-dithiobis-(2-nitrobenzoicacid) as the chromogenic agent. The molecular weight of the enzyme was determined as about 70 kDa monomer upon disulphide reduction by sodium dodecyl sulphate polyacrylamide gel electrophoresis. K m, V max, optimum pH and optimum temperature for acetylcholinesterase were found by means of graphics for acetylthiocholine iodide as the substrate. The optimum pH and optimum temperature of the acetylcholinesterase were determined to be 7.4 and 25–35 °C. The Michaelis–Menten constant (K m) for the hydrolysis of acetylthiocholine iodide was found to be 0.25 mM, and the V max was 0.090 μmol/mL/min. Maximum binding was achieved at 2 °C with pH 7.4 and an ionic strength of approximately 0.1 M. The capacity for the optimum condition was 0.07 mg protein/g gel for acetylcholinesterase.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Li, F., & Han, Z. (2002). Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover). Archives of Insect Biochemistry and Physiology, 51, 37–45.

    Article  CAS  Google Scholar 

  2. Eldefrawi, A. T. (1985). Acetylcholinesterases and anticholinesterases. In G. A. Kerkut & L. I. Gilbert (Eds.), Comprehensive insect physiology, biochemistry and pharmacology (pp. 115–130). Oxford: Pergamon.

    Google Scholar 

  3. Milatovic, D., & Dettbarn, W. D. (1996). Modification of acetylcholinesterase during adaptation to chronic, subacute paraoxon application in rat. Toxicology and Applied Pharmacology, 136, 20–28.

    Article  CAS  Google Scholar 

  4. Massoulie, J., & Bon, S. (1982). The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. Annual Review of Neuroscience, 5, 57–106.

    Article  CAS  Google Scholar 

  5. Silman, I., Giamberardino, L. D., Lyles, J., & Couraud, J. Y. (1979). Parallel regulation of acetylcholinesterase and pseudocholinesterase in normal, denervated and dystrophic chicken skeletal muscle. Nature, 280, 160–162.

    Article  CAS  Google Scholar 

  6. Ott, P. (1985). Membrane acetylcholinesterases-purification, molecular-properties and interactions with amphiphilic environments. Biochimica et Biophysica Acta, 822, 375–392.

    Article  CAS  Google Scholar 

  7. Rotundo, R. L. (1984). Asymmetric acetylcholinesterase is assembled in the golgi apparatus. Proceedings of the National Academy of Sciences, 91, 479–483.

    Article  Google Scholar 

  8. Chhajlani, V., Derr, D., Earles, B., Schmell, E., & August, T. (1989). Purification and partial amino acid sequence analysis of human erythrocyte acetylcholinesterase. FEBS Letters, 247, 279–282.

    Article  CAS  Google Scholar 

  9. Rosenberry, T. L., & Scoggin, D. M. (1984). Structure of human erythrocyte acetylcholinesterase characterization of intersubunit disulfide bonding and detergent interaction. Journal of Biological Chemistry, 259, 5643–5652.

    CAS  Google Scholar 

  10. Dutta-Choudhary, T. A., & Rosenberry, T. L. (1984). Human erythrocyte acetylcholinesterase is an amphipathic protein whose short membrane-binding domain is removed by papain digestion. Journal of Biological Chemistry, 259, 5653–5660.

    Google Scholar 

  11. Ahmed, M., Rocha, J. B. T., Correa, M., Mazzanti, C. M., Zanin, R. F., Morsch, A. L. B., et al. (2006). Inhibition of two different cholinesterases by tacrine. Chemico-Biological Interactions, 162, 165–171.

    Article  CAS  Google Scholar 

  12. Piau, A., Nourhashemi, F., Hein, C., Caillaud, C., & Vellas, B. (2011). Progress in the development of new drugs in Alzheimer's disease. The Journal of Nutrition, Health & Aging, 15, 45–57.

    Article  CAS  Google Scholar 

  13. Hayour, H., Bouraiou, A., Bouacid, S., Berre, F., Carboni, B., Roisnel, T., et al. (2011). Synthesis and X-ray structures of new cycloalka[e]pyrano[2,3b]pyridinederivatives: novel tacrine analogues. Tetrahedron Letters, 52, 4868–4871.

    Article  CAS  Google Scholar 

  14. Jin, Q. H., Shi, Y. F., He, H. Y., Ng, K. K., Jiang, H., Yang, L., et al. (2002). Isolation of acetylcholinesterase from apoptotic human lung fibroblast cells by antibody affinity chromatography. Bio Techniques, 33, 92–97.

    Google Scholar 

  15. Keane, S., & Ryan, M. F. (1999). Purification, characterisation, and inhibition by monoterpenes of acetylcholinesterase from the waxmoth Galleria mellonella (L.). Insect Biochemistry and Molecular Biology, 29, 1097–1104.

    Article  CAS  Google Scholar 

  16. Talesa, V., Grauso, M., Principato, G. B., Giovannini, E., Norton, S. J., & Rosi, G. (1994). Presence of soluble tetrameric (blood) and membrane-bound dimeric forms of cholinesterase in the mollusk, Murex brandaris (Gastropoda: Neogastropoda). The Journal of Experimental Zoology, 270, 233–244.

    Article  CAS  Google Scholar 

  17. Carroll, R. T., Grimm, J. L., Hepburn, T. W., & Emmerling, M. R. (1995). Purification of acetylcholinesterase by tacrin affinity chromatography. Protein Expression and Purification, 6, 389–393.

    Article  CAS  Google Scholar 

  18. Marchot, P., Ravelli, R. B. G., Raves, M. L., Bourne, Y., Vellom, D. C., Kanter, I. J., et al. (1996). Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin. Protein Science, 5, 672–679.

    Article  CAS  Google Scholar 

  19. Taylor, P., & Radic, Z. (1994). The cholinesterases: from genes to proteins. Annual Review of Pharmacology and Toxicology, 3, 281–320.

    Google Scholar 

  20. Askar, K. A., Kudi, A. C., & Moody, A. J. (2011). Purification of soluble acetylcholinesterase from sheep liver by affinity chromatography. Applied Biochemistry and Biotechnology, 165, 336–346.

    Article  CAS  Google Scholar 

  21. Cuatrecasa, P. (1970). Protein purification by affinity chromatography. The Journal of Biological Chemistry, 245, 3059–3065.

    Google Scholar 

  22. Porath, J. (1968). Sweden: molecular sieving and adsorption. Nature, 218, 834–838.

    Article  CAS  Google Scholar 

  23. Hjerten, S. (1962). Chromatographic separation according to size of macromolecules and cell particles on columns of agarose suspensions. Archives of Biochemistry and Biophysics, 99, 466–475.

    Article  CAS  Google Scholar 

  24. Hunter, A. J., Murray, T. K., Jones, J. A., Cross, A. J., & Green, A. R. (1989). The cholinergic pharmacology of tetrahydroaminoacridine in vivo and in vitro. British Journal of Pharmacology, 98, 79–86.

    Article  CAS  Google Scholar 

  25. Arslan, O., Nalbantoglu, B., Demir, N., Ozdemir, H., & Kufrevioglu, O. I. (1996). A new method for the purification of carbonic anhydrase isoenzymes by affinity chromatography. Turkish Journal of Medical Sciences, 26, 163–166.

    CAS  Google Scholar 

  26. Rosenberry, T. L., Chen, J. F., Lee, M. M. L., Moulten, T. A., & Onigmann, P. (1981). Large scale isolation of human erythrocyte membranes by high volume molecular filtration. Journal of Biochemical and Biophysical Methods, 4, 39–48.

    Article  CAS  Google Scholar 

  27. Laemmli, D. K. (1975). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  Google Scholar 

  28. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–251.

    Article  CAS  Google Scholar 

  29. Ellman, G. L., Courtney, K. D., Andres, V., & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7, 88–95.

    Article  CAS  Google Scholar 

  30. Lineweaver, H., & Burk, D. (1934). The determination of enzyme dissociation constants. Journal of the American Chemical Society, 56, 658–666.

    Article  CAS  Google Scholar 

  31. Bukowska, B., & Hutnik, K. (2006). 2,4-D and MCPA and their derivatives: effect on the activity of membrane erythrocytes acetylcholinesterase (in vitro). Pesticide Biochemistry and Physiology, 85, 174–180.

    Article  CAS  Google Scholar 

  32. Boschetti, M., & Brodbeck, U. (1996). The membrane anchor of mammalian brain acetylcholinesterase consists of a single glycosylated protein of 22 kDa. FEBS Letters, 380, 133–136.

    Article  CAS  Google Scholar 

  33. Al-Jafari, A. A. (1993). Investigation of the reversible inhibition of camel (Camelus dromedaries) acetylcholinesterase by tetracaine. Comparative Biochemistry and Physiology, 105, 323–327.

    Article  CAS  Google Scholar 

  34. Al-Jafari, A. A., Al-Khwyter, F., Kamal, M. A., & Alhomida, A. S. (1996). Kinetics for camel (Camelus dromedarius) retina acetylcholinesterase inhibition by methotrexate in vitro. Japanese Journal of Pharmacology, 72, 49–55.

    Article  CAS  Google Scholar 

  35. Neville, L. F., Gnatt, A., Loewenstein, Y., & Soreq, H. (1990). Aspartate-70 to glycine substitution confers resistance to naturally occurring and synthetic anionic-site ligands on inovo produced human butyrylcholinesterase. Journal of Neuroscience Research, 27, 452–460.

    Article  CAS  Google Scholar 

  36. Al-Khwyter, F., Kamal, M. A., & Al-Jafari, A. A. (1996). The inhibitory effect of cyclophosphamide on camel retina acetylcholine-esterase activity. Toxicology Letters, 87, 69–76.

    Article  CAS  Google Scholar 

  37. Adewusi, E. A., Moodley, N., & Steenkamp, V. (2011). Antioxidant and acetylcholinesterase inhibitory activity of selected Southern African medicinal plants. South African Journal of Botany, 77, 638–644.

    Article  CAS  Google Scholar 

  38. Jin, Z., Yang, L., Liu, S. J., Wang, J., Li, S., Lin, H. Q., et al. (2010). Synthesis and biological evaluation of 3,6-diaryl-7H-thiazolo[3,2-b][1,2,4]triazin-7-one derivatives as acetylcholinesterase inhibitors. Archives of Pharmacal Research, 33, 1641–1649.

    Article  CAS  Google Scholar 

  39. Tusarova, I., Halamek, E., & Kobliha, Z. (1999). Study on reactivation of enzyme-inhibitor complexes by oximes using acetylcholine esterase inhibited by organophosphate chemical warfare agents. Enzyme and Microbial Technology, 25, 400–403.

    Article  CAS  Google Scholar 

  40. Al-Jafari, A. A., Kamal, M. A., Greig, N. H., Alhomida, A. S., & Perry, E. R. (1998). Kinetics of human erythrocyte acetlycholinesterase inhibition by a novel derivative of physostigmine: phenserine. Biochemical and Biophysical Research Communications, 248, 180–185.

    Article  CAS  Google Scholar 

  41. Shapiro, R., & Vallee, B. L. (1991). Interaction of human placental ribonucleic with placental ribonuclease inhibitor. Biochemistry, 30, 2246–2255.

    Article  CAS  Google Scholar 

  42. Segel, I. H. (1993). Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. New York: Wiley-Interscience.

    Google Scholar 

  43. Ozdemir, H., Aygul, I., & Kufrevioglu, O. I. (2001). Purification of lactoperoxidase from bovine milk and investigation of the kinetic properties. Preparative Biochemistry and Biotechnology, 31, 125–134.

    Article  CAS  Google Scholar 

  44. Berman, H. A., & Leonard, K. (1992). Interaction of tetrahydroaminoacridine with acetylcholinesterase and butyrylcholinesterase. Molecular Pharmacology, 41, 412–418.

    CAS  Google Scholar 

  45. Cheng, D. H., & Tang, X. C. (1998). Comparative studies of huperzine A, E2020, and tacrine on behavior and cholinesterase activity. Pharmacology Biochemistry and Behavior, 60, 377–386.

    Article  CAS  Google Scholar 

  46. Porcelli, F., Delfini, M., & Giudice, M. R. D. (1999). The kinetic inhibition of acetylcholinesterase from human erythrocyte by tacrine and some tacrine derivatives. Bioorganic Chemistry, 27, 197–205.

    Article  CAS  Google Scholar 

  47. Shin, K., Hayasawa, H., & Lönnerdal, B. (2001). Purification and quantification of lactoperoxidase in human milk with use of immunoadsorbents with antibodies against recombinant human lactoperoxidase. American Journal of Clinical Nutrition, 73, 984–989.

    CAS  Google Scholar 

  48. Nandini, K. E., & Rastogi, N. K. (2010). Single step purification of lactoperoxidase from whey involving reverse micelles-assisted extraction and its comparison with reverse micellar extraction. Biotechnology Progress, 26, 763–771.

    Article  CAS  Google Scholar 

  49. Guedes, R. N. C., Zhu, K. Y., Kambhampati, S., & Dover, B. (1998). Characterization of acetylcholinesterase purified from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae). Comparative Biochemistry and Physiology, 119, 205–210.

    CAS  Google Scholar 

  50. Zhu, K. Y., & Brindley, W. A. (1992). Enzymological and inhibitory properties of acetylcholinesterase purified from Lygus hesperu Knight (Hemiptera: Miridae). Insect Biochemistry and Molecular Biology, 22, 245–251.

    Article  CAS  Google Scholar 

  51. Forget, J., & Bocquene, G. (1999). Partial purification and enzymatic characterization of acetylcholinesterase from the intertidal marine copepod Tigriopus brevicornis. Comparative Biochemistry and Physiology, 123, 345–350.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, Faculty of Sciences, Ataturk University, 25240, Erzurum, Turkey

    Habibe Budak Kaya, Bilge Özcan & Hasan Ozdemir

  2. Department of Molecular Biology and Genetics, Faculty of Sciences, Ataturk University, 25240, Erzurum, Turkey

    Melda Şişecioğlu

Authors
  1. Habibe Budak Kaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bilge Özcan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Melda Şişecioğlu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hasan Ozdemir
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hasan Ozdemir.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kaya, H.B., Özcan, B., Şişecioğlu, M. et al. Purification of Acetylcholinesterase by 9-Amino-1,2,3,4-tetrahydroacridine from Human Erythrocytes. Appl Biochem Biotechnol 170, 198–209 (2013). https://doi.org/10.1007/s12010-013-0177-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-013-0177-3

Keywords

Search

Navigation