Summary
The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.
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Albuquerque, E.X., Sokoll, M.D., Sonesson, B., Thesieff, S.: Studies on the nature of the cholinergic receptor. Eur. J. Pharmacol. 4, 40–46 (1968)
Betz, W., Sakmann, B.: ‘Disjunction’ of frog neuromuscular synapses by treatment with proteolytic enzymes. Nature (New Biol.) 232, 94–95 (1971)
Betz, W., Sakmann, B.: Effects of proteolytic enzymes on function and structure of frog neuromuscular junction. J. Physiol. 230, 673–688 (1973)
Birks, R.I., Brown, L.M.: A method for locating the cholinesterase of a mammalian myoneural junction by electron microscopy. J. Physiol. (London) 152, 5–7P (1960)
Bon, S., Cartaud, J., Massoluié, J.: The dependence of acetylcholinesterase aggregation at low ionic strength upon a polyanionic component. Eur. J. Biochem. 85, 1–14 (1978)
Brzin, M., Zajiček, J.: Quantitative determination of cholinesterase activity in individual end-plates of normal and denervated gastrocnemius muscle. Nature 181, 626 (1958)
Brzin, M., Pucihar, S.: Iodide, thiocyanate and cyanide ions as capturing reagents in one-step copper thiocholine method for cytochemical localization of cholinesterase activity. Histochemistry 48, 283–292 (1976)
Brzin, M.: Ultrastructural localization of acetylcholinesterase activity in relation to membranes of neuromuscular junction. Period. biol. 80, 63–68 (1978)
Brzin, M., Kiauta, T.: Cholinesterases of the motor endplate of the rat diaphragm. In: Progress in Brain Research, Vol. 49: The Cholinergic Synapse. S. Tuček (ed.), p. 311. Amsterdam: Elsevier-North Holland 1979
Couteaux, R., Taxi, J.: Recherches histochimiques sur la distribution des activités cholinesterasiques au niveau de la synapse myoneurale. Arch. Anat. micr. Morph. exp. 41, 352–392 (1952)
Davis, R., Koelle, G.B.: Electron microscopic localization of acetylcholinesterase and nonspecific cholinesterase at the neuromuscular junction by the gold-thiocholine and gold-thiolacetic acid methods. J. Cell Biol. 34, 157–171 (1967)
Dudai, Y., Silman, I.: The effects of solubilization procedures on the release and molecular state of acetylcholinesterase from electric organ tissue. J. Neurochem. 23, 1177–1187 (1974)
Friedenberg, R.M., Seligman, A.M.: Acetylcholinesterase at the myoneural junction: cytochemical ultrastructure and some biochemical considerations. J. Histochem. Cytochem. 20, 771–792 (1972)
Hall, Z.W., Kelly, R.B.: Enzymatic detachment of endplate acetylcholinesterase from muscle. Nature (New Biol.) 232, 62–63 (1971)
Jonecko, A.: Über die PAS-Substanz an der motorischen Endplatte und myotendinösen Junktion. Acta Histochem. (Jena) 36, 113–118 (1970)
Koelle, G.B., Friedenwald, J.S.: A histochemical method for localizing cholinesterase activity. Proc. Soc. Exp. Biol. Med. 70, 617–622 (1949)
Kono, T., Kakuma, F., Homa, M., Fukuda, S.: The electron-microscopic structure and chemical composition of the isolated sarcolemma of the rat sketetal muscle cell. Biochim. Biophys. Acta 88, 155–176 (1964)
Lehrer, G.M., Ornstein, L.A.: A diazo coupling method for the electron microscopic localization of cholinesterase. J. Biophys. biochem. Cytol. 6, 390–406 (1959)
Lewis, M.K., Eldefrawi, M.E.: A simple, rapid and quantitative radiometric assay of acetylcholinesterase. Anal. Biochem. 57, 588–592 (1974)
Liu, A.Y.C., Mittag, T.W.: Membrane-bound acetylcholinesterase: comparison of enzymes in sarcoplasmic reticulum and sarcolemma from striated muscle. Mol. Pharmacol. 10, 283–292 (1974)
Marnay, A., Nachmansohn, D.: Cholinesterase in voluntary muscle. J. Physiol. 92, 37–47 (1938)
Massoulié, J., Rieger, F., Tsuji, S.: Solubilisation de l'acétylcholinestérase des organes électriques de gymnote. Action de la trypsine. Eur. J. Biochem. 14, 430–439 (1970)
McMahan, U.J., Spitzer, N.C., Peper, K.: Visual identification of nerve terminals in living isolated skeletal muscle. Proc. R. Soc. Lond. B. 181, 421–430 (1972)
McMahan, U.J., Sanes, J.R., Marshall, L.M.: Cholinesterase is associated with the basal lamina at the neuromuscular junction. Nature 271, 172–174 (1978)
Rambourg, A., Leblond, C.P.: Electron microscope observations on the carbohydrate-rich cell coat present at the surface of cells in the rat. J. Cell Biol. 32, 27–53 (1967)
Rash, J.E., Elisman, M.N.: Studies of excitable membranes 1. Neuromuscular specialization of the neuromuscular junction and the nonjunctional sarcolemma. J. Cell Biol. 63, 567–586 (1974)
Seifter, S., Harper, E.: The collagenases. In: The enzymes. P.D. Boyer (ed.), pp. 649–697. New York and London: Academic Press 1971
Sketelj, J., Brzin, M.: Increase in the apparent AChE activity in the mouse diaphragm induced by proteolytic treatment. J. Neurochem. 29, 109–114 (1977)
Taylor, P., Lwebuga-Mukasa, J., Berman, H., Lappi, S.: Spectroscopic studies of ligand interactions with acetylcholinesterase. Croatica Chemica Acta 47, 251–263 (1975)
Tennyson, V.M., Kremzner, L.T., Brzin, M.: Electron microscopic-cytochemical and biochemical studies of acetylcholinesterase activity in denervated muscle of rabbits. J. Neuropath. Exp. Neurol. 36, 245–275 (1977)
Tennyson, V.M., Miranda, A., Kremzner, L.T.: Electron-microscopic, cytochemical and biochemical studies of acetylcholinesterase and butyrylcholinesterase activity in muscles of dystrophic mice. J. Neurol. Sci. 25, 309–332 (1975)
Ulbrecht, G., Kruckenberg, P.: Acetylcholinesterase in the sarcoplasmatic reticulum of skeletal muscle. Nature 206, 305–306 (1965)
Vandenburgh, H.H., Sheff, M.F., Zacks, S.I.: Chemical composition of isolated rat skeletal sarcolemma. J. Membrane Biol. 17, 1–12 (1974)
Zacks, S.I., Saito, A., Sheff, M.F.: Cytochemical properties of the external lamina of myofibers and neuromuscular junctions. In: The striated muscle. C.M. Pearson, F.K. Mostafi (eds.), pp. 123–143. Baltimore: The Williams and Wilkins Comp. 1973a
Zacks, S.I., Sheff, M.F., Saito, A.: Structure and staining characteristics of myofiber external lamina. J. Histochem. Cytochem. 21, 703–714 (1973b)
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Sketelj, J., Brzin, M. Attachment of acetylcholinesterase to structures of the motor endplate. Histochemistry 61, 239–248 (1979). https://doi.org/10.1007/BF00508444
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DOI: https://doi.org/10.1007/BF00508444