Summary
InTorpedo electric organ, much of the acetylcholinesterase (AChE) is a globular dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and selectively solubilized by a bacterial phosphatidylinositol-specific phospholipase C. While the structure of this form of the enzyme is well-established, the ultrastructural localization of G2-AChE is still unclear. Selective solubilization with phosphatidylinositol-specific phospholipase C was, therefore, combined with immunocytochemistry at the electron microscope level, in order to localize G2-AChE in electric organ ofTorpedo ocellata. Thin sections of electric organ were labelled with antibodies raised againstTorpedo AChE, followed by gold-conjugated second antibodies, before or after exposure to the phospholipase. For comparison, the location of AChE was examined using histochemical methods. We show that (1) immunolabelling is concentrated in the synaptic clefts between nerve terminals and the innervated face of the electrocyte; (2) this labelling co-localizes with AChE histochemical reaction products; and (3) prior exposure to the phospholipase causes a decrease in AChE-associated labelling. Quantitative analysis of immunolabelling in the synaptic clefts shows that the phospholipase treatment had reduced primary labelling at or adjacent to the presynaptic membrane. Together with our earlier biochemical and immunofluorescent evidence, these results support our previous assignment of a neuronal and synaptic localization for G2-AChE inTorpedo electric organ.
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Abramson, S. N., Ellisman, M. H., Deerinck, T. J., Maulet, Y., Gentry, M. K., Doctor, B. P. &Taylor, P. (1989) Differences in structure and distribution of the molecular forms of acetylcholinesterase.Journal of Cell Biology 108, 2301–11.
Anglister, L. (1991) Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber.Journal of Cell Biology 115, 755–64.
Anglister, L. &McMahan, U. J. (1984) Extracellular matrix components involved in neuromuscular transmission and regeneration. InBasement Membranes and Cell Movement (edited byPorter, R. &Whelan, R.) pp. 161–78. London: Pitman.
Bartfeld, D. &Fuchs, S. (1979) Fractionation of antibodies to acetylcholine receptor according to antigenic specificity.FEBS Letters 105, 303–6.
Bloom, F. E. &Barrnett, R. J. (1966) Fine structural localization of acetylcholinesterase in electroplaque of the electric eel.Journal of Cell Biology 29, 475–95.
Brimijoin, S. &Rakonczay, Z. (1986) Immunology and molecular biology of the cholinesterases: current results and prospects.International Review of Neurobiology 28, 363–410.
Brown, W. J. &Farquhar, M. G. (1984) The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis golgi cisternae.Cell 36, 295–307.
Doctor, B. P., Camp, S., Gentry, M. K., Taylor, S. S. &Taylor, P. (1983) Antigenic and structural differences in the catalytic subunits of the molecular forms of acetylcholinesterase.Proceedings of the National Academy of Science (USA) 80, 5767–71.
Eichler, J., Silman, I., Gentry, M. K. &Anglister, L. (1990) Immunocytochemical localization of phosphatidy-linositol-anchored acetylcholinesterase in excitable tissues ofTorpedo ocellata.Molecular Brain Research 8, 213–18.
Federov, V. V. (1981) Postsynaptic end plate ionic currents in fast and slow muscle fibres of chickens, effect of cholinesterase inhibition.Neurophysiology 13, 396–7.
Futerman, A. H., Low, M. G. &Silman, I. (1983) A hydrophobic dinner of acetylcholinesterase fromTorpedo californica electric organ is solubilized by phosphatidylinositol-specific phospholipase C.Neuroscience Letters 40, 85–9.
Futerman, A. H., Fiorini, R. M., Roth, E., Low, M. G. &Silman, I. (1985a) Physicochemical behaviour and structural characteristics of membrane-bound acetylcholinesterase fromTorpedo electric organ.Biochemical Journal 226, 369–77.
Futerman, A. H., Low, M. G., Michaelson, D. M. &Silman, I. (1985b) Solubilization of membrane-bound acetylcholinesterase by a phosphatidylinositol-specific phospholipase C.Journal of Neurochemistry 45, 1487–94.
Futerman, A. H., Raviv, D., Michaelson, D. M. &Silman, I. (1987) Differential susceptibility to phosphatidy-linositol-specific phospholipase C of acetylcholinesterase in excitable tissues of embryonic and adultTorpedo ocellata.Molecular Brain Research 2, 105–12.
Jedrzejczyk, J., Silman, I., Lai, J. &Barnard, E. A. (1984) Molecular forms of acetylcholinesterase in synaptic and extrasynaptic regions of avian tonic muscle.Neuroscience Letters 46, 283–9.
Johnson, C. D. &Russell, R. L. (1975) A rapid simple radiometric assay for cholinesterase suitable for multiple determinations.Analytical Biochemistry 64, 229–38.
Karnovsky, M. J. &Roots, L. (1964) A “direct-coloring” thiocholine method for cholinesterases.Journal of Histochemistry and Cytochemistry 12, 219–21.
Kushner, P. D., Stephenson, D. T., Sternberg, H. &Weber, R. (1987) Monoclonal antibodyTor 23 recognizes a determinant of a presynaptic acetylcholinesterase.Journal of Neurochemistry 48, 1942–53.
Laskey, R. A. &Mills, A. D. (1975) Quantitative film detection of3H and14C in polyacrylamide gels by fluorography.European Journal of Biochemistry 56, 335–41.
Li, Z. Y. &Bon, C. (1983) Presence of a membrane-bound acetylcholinesterase form in a preparation of nerve endings fromTorpedo marmorata electric organ.Journal of Neurochemistry 40, 338–49.
Low, M. G., Ferguson, M. A. J., Futerman, A. H. &Silman, I. (1986) Covalently attached phosphatidylinositol as a hydrophobic anchor for membrane proteins.Trends in Biochemical Sciences 11, 212–15.
Low, M. G., Stiernberg, J., Waneck, G. L., Flavell, R. A. &Kincade, P. W. (1988) Cell-specific heterogeneity in sensitivity of phosphatidylinositol-anchored membrane antigens to release by phospholipase C.Journal of Immunological Methods 113, 101–11.
Mailly, P., YounÈS-chenoufi, A. B. &Bon, S. (1989) The monoclonal antibodies Elec-39, HNK-1 and NC-1 recognize structures in the nervous system and muscles of vertebrates.Neurochemistry International 15, 517–30.
MassouliÉ, J. &Toutant, J. P. (1988) Vertebrate cholinesterases: structure and types of interaction. InHandbook of Experimental Pharmacology, vol. 86 (edited byWhittaker, V. P.) pp. 167–224. Berlin: Springer-Verlag.
Matthews-Bellinger, J. &Salpeter, M. M. (1978) Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications.Journal of Physiology 279, 197–213.
Mclean, I. W. &Nakane, P. K. (1974) Periodate-lysineparaformaldehyde fixative. A new fixative for immuno-electron microscopy.Journal of Histochemistry and Cytochemistry 22, 1077–83.
Morel, N. &Dreyfus, P. (1982) Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane inTorpedo electric organ.Neurochemistry International 4, 283–8.
Sealock, R. &Kavookjian, A. (1980) Postsynaptic distribution of acetylcholine receptors in electroplax of the Torpedine rayNarcine brasiliensis.Brain Research 190, 81–93.
Sealock, R., Wray, B. &Froehner, S. C. (1984) Ultrastructural localization of the M' 43,000 protein and the acetylcholine receptor inTorpedo postsynaptic membranes using monoclonal antibodies.Journal of Cell Biology 98, 2239–44.
Silman, I., Lyles, J. M. &Barnard, E. A. (1978) Intrinsic forms of acetylcholinesterase in skeletal muscle.FEBS Letters 94, 166–70.
Silman, I. &Futerman, A. H. (1987) Modes of attachment of acetylcholinesterase to the surface membrane.European Journal of Biochemistry 170, 11–22.
Stirling, J. W. (1990) Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques.Journal of Histochemistry and Cytochemistry 38, 145–57.
Towbin, H. &Gordon, J. (1984) Immunoblotting and dot immunobinding-current status and outlook.Journal of Immunological Methods 72, 313–40.
Tsuji, S. (1974) On the chemical basis of thiocholine methods for demonstration of acetylcholinesterase activities.Histochemistry 42, 99–110.
Vigny, M., Gisiger, V. &MassouliÉ, J. (1978) “Non-specific” cholinesterase and acetylcholinesterase in rat tissues: molecular forms, structural and catalytic properties, and significance of the two enzyme systems.Proceedings of the National Academy of Science (USA) 75, 2588–92.
Witzemann, V. &Boustead, C. (1981) Distribution of acetylcholinesterase molecular forms in brain, nerve and muscle tissue ofTorpedo marmorata.Neuroscience Letters 26, 313–18.
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Eichler, J., Silman, I. & Anglister, L. G2-Acetylcholinesterase is presynaptically localized inTorpedo electric organ. J Neurocytol 21, 707–716 (1992). https://doi.org/10.1007/BF01181586
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DOI: https://doi.org/10.1007/BF01181586