Abstract
Crotoxin is a neurotoxic phospholipase A2 capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin withN-hydroxysuccinimidyl-4-azidobenzoate and with Na[125I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin,Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of about 10−8 M. Mojave toxin, caudoxin, notexin,Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while β-bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.
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Aird, S. D., Kaiser, I. I., Lewis, R. V., and Kruggel, W. G. (1985).Biochemistry 24, 7054–7058.
Aird, S. D., Kaiser, I. I., Lewis, R. V., and Kruggel, W. G. (1986).Biochemistry 249, 296–300.
Bon, C., Changeux, J. P., Jeng, T. W., and Fraenkel-Conrat, H. (1979).Eur. J. Biochem. 99, 471–481.
Booth, A. G., and Kenny, A. J. (1976).J. Cell Sci. 21, 449–463.
Breithaupt, H. (1976).Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 271.
Buckland, B. R., Howell, R. D., Richards, C. R., and Smith, B. (1985).Biochem. J. 225, 753–760.
Cate, R. L., and Bieber, A. L. (1978).Arch. Biochem. Biophys. 189, 397.
Fohlman, J., Eaker, D., Karlsson, E., and Thesleff, S. (1976).Eur. J. Biochem. 68, 457–469.
Hanley, M. R., Eterovic, V. A., Hawkes, S. P., Hebert, A. J., and Bennett, E. L. (1977).Biochemistry 16, 5840–5849.
Hawgood, B. J., and Smith, J. W. (1977).Br. J. Pharmacol. 61, 597–606.
Hazum, E. (1983).Methods Enzymol. 103, 58–71.
Hendon, R. A., and Fraenkel-Conrat, H. (1971).Proc. Natl. Acad. Sci. USA 68, 1560–1563.
Hendon, R. A., and Fraendel-Conrat, H. (1976).Toxicon 14, 285–289.
Howard, B. C., and Gundersen, C. G., Jr. (1980).Annu. Rev. Pharmacol. Toxicol. 20, 307–336.
Hunter, W. M., and Greenwood, F. C. (1962).Nature (London)194, 495–496.
Iyengar, R., and Herberg, J. T. (1984).J. Biol. Chem. 259, 5222–5229.
Ji, T. H. (1977).J. Biol. Chem. 252, 1566–1570.
Lo, T. B., Chen, Y. H., and Lee, C. Y. (1966).J. Chinese Chem. Soc. 13, 25.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951).J. Biol. Chem. 193, 265–275.
Neville, D. M. (1960).J. Biophys. Biochem. Cytol. 8, 413.
Neville, D. M., Jr. (1971).J. Biol. Chem. 246, 6328–6334.
Othman, I. B., Spokes, J. W., and Dolly, J. O. (1982).Eur. J. Biochem. 128, 267–276.
Rehm, H., and Betz, H. (1982).J. Biol. Chem. 257, 10015–10022.
Rehm, H., and Betz, H. (1983).EMBO J. 2, 1119–1122.
Rubsamen, K., Breithaupt, H., and Habermann, E. (1971).Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmakol. 270, 274–288.
Slotta, K., and Fraenkel-Conrat, H. (1938).Ber. Dtsch. Chem. Ges. 71, 1076–1081.
Steck, T. L., Weinstein, R. S., Strauss, J. H., and Wallach, D. F. H. (1970).Science 168, 255.
Tzeng, M.-C. (1983).Anal. Biochem. 128, 412–414.
Tzeng, M.-C., Hseu, M. J., Yang, J. H., and Guillory, R. J. (1986).J. Protein Chem. 5, 221–228.
Viljeon, C. C., Botes, D. P., and Kruger, H. (1982).Toxicon 20, 715–737.
Vita-Brazil, O. (1972).J. Formosan Med. Assoc. 71, 394–400.
Whittaker, V. P. (1959).Biochem. J. 72, 694–706.
Wong, M. M., Robertson, N. P., and Horowitz, J. (1978).Biochem. Biophys. Res. Commun. 84, 158–165.
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On leave from Department of Biochemistry and Biophysics, University of Hawaii School of Medicine, Honolulu, Hawaii.
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Hseu, M.J., Guillory, R.J. & Tzeng, MC. Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling. J Bioenerg Biomembr 22, 39–50 (1990). https://doi.org/10.1007/BF00762844
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DOI: https://doi.org/10.1007/BF00762844