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. 1989 Jul 15;261(2):569–573. doi: 10.1042/bj2610569

A study of the mechanism by which some amphiphilic drugs affect human erythrocyte acetylcholinesterase activity.

A Spinedi 1, L Pacini 1, P Luly 1
PMCID: PMC1138862  PMID: 2775233

Abstract

The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase.

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Selected References

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  1. Barton P. L., Futerman A. H., Silman I. Arrhenius plots of acetylcholinesterase activity in mammalian erythrocytes and in Torpedo electric organ. Effect of solubilization by proteinases and by a phosphatidylinositol-specific phospholipase C. Biochem J. 1985 Oct 1;231(1):237–240. doi: 10.1042/bj2310237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bloj B., Galo M. G., Morero R. D., Farias R. N. Kinetic modifications of the acetylcholinesterase and (Ca2+ + Mg2+)-ATPase in rat erythrocytes by cholesterol feeding. J Nutr. 1976 Dec;106(12):1827–1834. doi: 10.1093/jn/106.12.1827. [DOI] [PubMed] [Google Scholar]
  3. Bloj B., Morero R. D., Farías R. N. Effect of essential fatty acid deficiency on the arrhenius plot of acetylcholinesterase from rat erythrocytes. J Nutr. 1974 Oct;104(10):1265–1272. doi: 10.1093/jn/104.10.1265. [DOI] [PubMed] [Google Scholar]
  4. CHEN R. F., BOWMAN R. L. FLUORESCENCE POLARIZATION: MEASUREMENT WITH ULTRAVIOLET-POLARIZING FILTERS IN A SPECTROPHOTOFLUOROMETER. Science. 1965 Feb 12;147(3659):729–732. doi: 10.1126/science.147.3659.729. [DOI] [PubMed] [Google Scholar]
  5. Deliconstantinos G., Tsakiris S. Differential effect of anionic and cationic drugs on the synaptosome-associated acetylcholinesterase activity of dog brain. Biochem J. 1985 Jul 1;229(1):81–86. doi: 10.1042/bj2290081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donner M., Stoltz J. F. Comparative study on fluorescent probes distributed in human erythrocytes and platelets. Biorheology. 1985;22(5):385–397. doi: 10.3233/bir-1985-22503. [DOI] [PubMed] [Google Scholar]
  7. ELLMAN G. L., COURTNEY K. D., ANDRES V., Jr, FEATHER-STONE R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961 Jul;7:88–95. doi: 10.1016/0006-2952(61)90145-9. [DOI] [PubMed] [Google Scholar]
  8. Foot M., Cruz T. F., Clandinin M. T. Effect of dietary lipid on synaptosomal acetylcholinesterase activity. Biochem J. 1983 May 1;211(2):507–509. doi: 10.1042/bj2110507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frenkel E. J., Roelofsen B., Brodbeck U., van Deenen L. L., Ott P. Lipid-protein interactions in human erythrocyte-membrane acetylcholinesterase. Modulation of enzyme activity by lipids. Eur J Biochem. 1980 Aug;109(2):377–382. doi: 10.1111/j.1432-1033.1980.tb04804.x. [DOI] [PubMed] [Google Scholar]
  10. Futerman A. H., Low M. G., Michaelson D. M., Silman I. Solubilization of membrane-bound acetylcholinesterase by a phosphatidylinositol-specific phospholipase C. J Neurochem. 1985 Nov;45(5):1487–1494. doi: 10.1111/j.1471-4159.1985.tb07217.x. [DOI] [PubMed] [Google Scholar]
  11. Haas R., Brandt P. T., Knight J., Rosenberry T. L. Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase. Biochemistry. 1986 Jun 3;25(11):3098–3105. doi: 10.1021/bi00359a005. [DOI] [PubMed] [Google Scholar]
  12. Inestrosa N. C., Roberts W. L., Marshall T. L., Rosenberry T. L. Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues. J Biol Chem. 1987 Apr 5;262(10):4441–4444. [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Low M. G., Futerman A. H., Ackermann K. E., Sherman W. R., Silman I. Removal of covalently bound inositol from Torpedo acetylcholinesterase and mammalian alkaline phosphatases by deamination with nitrous acid. Evidence for a common membrane-anchoring structure. Biochem J. 1987 Jan 15;241(2):615–619. doi: 10.1042/bj2410615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Massoulié J., Bon S. The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. Annu Rev Neurosci. 1982;5:57–106. doi: 10.1146/annurev.ne.05.030182.000421. [DOI] [PubMed] [Google Scholar]
  16. Mazzanti L., Pastuszko A., Lenaz G. Effects of ketamine anesthesia on rat-brain membranes: fluidity changes and kinetics of acetylcholinesterase. Biochim Biophys Acta. 1986 Sep 25;861(1):105–110. doi: 10.1016/0005-2736(86)90376-7. [DOI] [PubMed] [Google Scholar]
  17. Ott P. Membrane acetylcholinesterases: purification, molecular properties and interactions with amphiphilic environments. Biochim Biophys Acta. 1985 Dec 9;822(3-4):375–392. doi: 10.1016/0304-4157(85)90016-4. [DOI] [PubMed] [Google Scholar]
  18. Roberts W. L., Rosenberry T. L. Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase. Biochemistry. 1986 Jun 3;25(11):3091–3098. doi: 10.1021/bi00359a004. [DOI] [PubMed] [Google Scholar]
  19. Shinitzky M., Barenholz Y. Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim Biophys Acta. 1978 Dec 15;515(4):367–394. doi: 10.1016/0304-4157(78)90010-2. [DOI] [PubMed] [Google Scholar]
  20. Sidek H. M., Nyquist-Battie C., Vanderkooi G. Inhibition of synaptosomal enzymes by local anesthetics. Biochim Biophys Acta. 1984 Sep 7;801(1):26–31. doi: 10.1016/0304-4165(84)90208-3. [DOI] [PubMed] [Google Scholar]
  21. Spinedi A., Rufini S., Luly P., Farias R. N. The temperature-dependence of human erythrocyte acetylcholinesterase activity is not affected by membrane cholesterol enrichment. Biochem J. 1988 Oct 15;255(2):547–551. [PMC free article] [PubMed] [Google Scholar]
  22. Wiedmer T., Di Francesco C., Brodbeck U. Effects of amphiphiles on structure and activity of human erythrocyte membrane acetylcholinesterase. Eur J Biochem. 1979 Dec;102(1):59–64. doi: 10.1111/j.1432-1033.1979.tb06262.x. [DOI] [PubMed] [Google Scholar]

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