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Covalent Affinity Chromatography of Acetylcholinesterase

  • W. G. Soucie
  • H. F. Voss
  • I. B. Wilson

Abstract

Active site directed inhibitors of the active serine residue of the catalytic center of acetylcholinesterase can be immobilized by covalently bonding them to derivatized agarose gels. If the immobilized inhibitor is an “irreversible” inhibitor, then not only is there a high degree of specificity for acetylcholinesterase, but there is also a certain amount of stability achieved because the enzyme will be covalently bonded to the gel. These considerations prompted Ashani and Wilson (1) to design an affinity gel that had as its ligand a methylphosphonate derivative which is an active site directed irreversible inhibitor of acetylcholinesterase. Eq. 1 demonstrates the general features of the binding,where E is acetylcholinesterase and the -OH of E-OH is the hydroxyl group of the active serine at the esteratic site; the inhibitor is aminoethyl p-nitrophenylmethylphosphonate, R is succinyldiaminopentylagarose gel, and p-nitrophenol is the leaving group. It has been demonstrated that this immobilized methylphosphonate has good specificity for acetylcholinesterase from electric organs ofElectrophorus eZectricus,Torpedo californica, as well as for α-chymotrypsin (1, 4–7).

Keywords

Electric Organ Trapping Efficiency Diphenyl Phosphoryl Organic Solvent System Esteratic Site 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    ASHANI, Y. and Wilson, I.B.Biochim. Biophys. Acta, 276: 317, 1972.CrossRefGoogle Scholar
  2. 2.
    WILSON, I.B. and Bergmann, F.J. Biol. Chem.185: 479, 1950.Google Scholar
  3. 3.
    KOELLE, G.B. (Ed) “Cholinesterases and Anti-cholinesterase Agents”, Vol. 15 of Handbuch der Experimentellen Pharmakologie, Springer Verlag, Berlin, 1963.Google Scholar
  4. 4.
    VOSS, H.F. Ph.D. Thesis. University of Colorado, Boulder, Colorado, 1974, p. 131.Google Scholar
  5. 5.
    VOSS, H.F., Ashani, Y. AND Wilson, I.B.Adv. Exp. Med. Biol.42: 75, 1974.Google Scholar
  6. 6.
    VOSS, H.F., Ashani, Y. and Wilson, I.B.Meth. Enzymol. 34:581,1974. Google Scholar
  7. 7.
    SOUCIE, W.G. Unpublished data.Google Scholar
  8. 8.
    HEILBROON, E.Act. Chem. Scand. 18:2410,1964. Google Scholar
  9. 9.
    FROEDE, H.C. and Wilson, I.B.J. Am. Chem. Soc. 95:1987,1973. Google Scholar
  10. 10.
    WILSON, I.B. and Ginsburg, S.Biochim. Biophys. Acta 18: 168,1955. Google Scholar
  11. 11.
    CHILDS, A.F., Davies, D.R., Green, A.L. and Rutland, J.P.Brit. J. Pharm. 10:462,1955. Google Scholar
  12. 12.
    GINSBURD, S. and Wilson, I.B.J. Am. Chem. Soc.79:481,1957. Google Scholar
  13. 13.
    WILSON, I.B. and Quan, C.Arch. Biochem. Biophys. 73:131,1958. Google Scholar
  14. 14.
    WINS, P. and Wilson, I.B.Biochim. Biophys. Acta,334:137,1974. Google Scholar
  15. 15.
    MAGLOTHIN, J.A. Ph.D. Thesis, University of Colorado, Boulder, Colorado,1974,p.127. Google Scholar
  16. 16.
    MAGLOTHIN, J.A., Wins, P. and Wilson, I.B.Biochim. Biophys. Acta 403:370,1975. Google Scholar
  17. 17.
    COHEN, J.A., OOSTERBAAN, R.A., JANG, H.S. and BERENDS, F.J. Cell Comp. Physiol.,54,Suppl.1: 231,1959. Google Scholar
  18. 18.
    BERENDS, F., POSTHUMUS, C.H., VANDER SLUYS, I. DEIERKAUF.Biochim. Biophys. Acta 34:576,1959. Google Scholar
  19. 19.
    SMITH, T.E. & USDIN, E.Biochemistry 5:2914,1966. Google Scholar
  20. 20.
    KIRBY, A.J. & YOUNAS, M.J. Chem. Soc. (B):1165,1970. Google Scholar
  21. 21.
    SVENSSON, B.Lett. 29:167,1973. Google Scholar
  22. 22.
    ARAKI, C. Bull Chem. Soc. Japan,29:543,1956. Google Scholar
  23. 23.
    Tessor, C.K., Fisch, H.-V. and Schwyzer, R.FEBS Lett. 23:56,1972. Google Scholar
  24. 24.
    Yong, M.S.Science,182:157,1973. Google Scholar
  25. 25.
    Patrick, J., Lindstrom, J., Culp, B. and Mcmillan, J.Proc. Nat. Acad. Sci. U.S.A.,70:3334,1973. Google Scholar
  26. 26.
    March, S.C., Parikh, I. and Cuatrecasas, P.Adv. Expt. Med. Biol. 42:3,1974. Google Scholar
  27. 27.
    Rosenberry, T.L., Chang, H.W. and Chen, Y.T.J. Biol. Chem. 193: 265,1951. Google Scholar
  28. 28.
    LOWRY, O.H., ROSENBROUGH, N.J., FARR, A.L. & RANDALL, R.J.J. Biol. Chem. 193:265,1951. Google Scholar
  29. 29.
    TANFORD, C. “Physical Chemistry of Macromolecules” John Wiley and Sons, New York,1961. Google Scholar
  30. 30.
    DUDAI, Y., SILMAN, I, SHINITSKY, M. & BLUMBERG, S.Proc. Nat. Acad. Sci. U.S.A. 69:2400,1972. Google Scholar
  31. 31.
    BON, S. & REIGER, F.FEBS Lett. 53: 282,1975. Google Scholar
  32. 32.
    REIGER, F., BON, S., MASSOULIE, J. & CARTAUD, J.Eur. J. Biochem. 34:539,1973. Google Scholar
  33. 33.
    DUDAI, Y., HERZBERG, M. & SILMAN, I.Proc. Nat. Acad. Sci. U.S.A. 70:2473,1973. Google Scholar

Copyright information

© Springer Science+Business Media New York 1978

Authors and Affiliations

  • W. G. Soucie
    • 1
  • H. F. Voss
    • 2
  • I. B. Wilson
    • 1
  1. 1.Chemistry Dept.University of ColoradoBoulderUSA
  2. 2.Chemistry Dept.University of California San DiegoLa JollaUSA

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