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. 1991 Dec 15;280(Pt 3):739–744. doi: 10.1042/bj2800739

Dissociation of lethal toxicity and enzymic activity of notexin from Notechis scutatus scutatus (Australian-tiger-snake) venom by modification of tyrosine residues.

C C Yang 1, L S Chang 1
PMCID: PMC1130516  PMID: 1764038

Abstract

Notexin from Notechis scutatus scutatus snake venom was subjected to tyrosine modification with p-nitrobenzenesulphonyl fluoride (NBSF), and four modified derivatives were separated by h.p.l.c. The results of amino acid analysis and sequence determination revealed that only Tyr-7, Tyr-70 and Tyr-77 were modified in notexin. Modification of Tyr-7 resulted in decreases in lethal toxicity and enzymic activity by 70.2% and 22.7% respectively. Conversely, modification of Tyr-77 caused a 1.8-fold increase in enzymic activity, in contrast with the loss of 52.5% of lethality. A drastic decrease in lethal toxicity was observed when both Tyr-7 and Tyr-70 were modified, whereas the enzymic activity decreased by only 35.8%. Likewise, the derivative in which Tyr-7 and Tyr-77 were modified retained 44.4% of enzymic activity, but showed a marked decrease in lethal toxicity. It is obvious that modification of tyrosine residues causes a decrease in lethal toxicity of notexin, which does not directly correlate with the change in enzymic activity. On the other hand, the antigenicity of NBS derivatives remained unchanged. The modified derivatives retained their affinity for Ca2+, indicating that the modified tyrosine residues did not participate in Ca2+ binding. These results indicate that modification of tyrosine residues can differentially influence the enzymic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one being responsible for the catalytic activity and the other associated with its lethal effect.

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Selected References

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  1. Brunie S., Bolin J., Gewirth D., Sigler P. B. The refined crystal structure of dimeric phospholipase A2 at 2.5 A. Access to a shielded catalytic center. J Biol Chem. 1985 Aug 15;260(17):9742–9749. [PubMed] [Google Scholar]
  2. Chang C. C. Neurotoxins with phospholipase A2 activity in snake venoms. Proc Natl Sci Counc Repub China B. 1985 Apr;9(2):126–142. [PubMed] [Google Scholar]
  3. Chang C. C., Su M. J. Mutual potentiation, at nerve terminals, between toxins from snake venoms which contain phospholipase A activity: beta-bungarotoxin, crotoxin, taipoxin. Toxicon. 1980;18(5-6):641–648. doi: 10.1016/0041-0101(80)90092-6. [DOI] [PubMed] [Google Scholar]
  4. Chang C. C., Su M. J. Presynaptic toxicity of the histidine-modified, phospholipase A2-inactive, beta-bungarotoxin, crotoxin and notexin. Toxicon. 1982;20(5):895–905. doi: 10.1016/0041-0101(82)90077-0. [DOI] [PubMed] [Google Scholar]
  5. Chang L. S., Yang C. C. Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait). J Protein Chem. 1988 Dec;7(6):713–727. doi: 10.1007/BF01025579. [DOI] [PubMed] [Google Scholar]
  6. Chwetzoff S., Mollier P., Bouet F., Rowan E. G., Harvey A. L., Ménez A. On the purification of notexin. Isolation of a single amino acid variant from the venom of Notechis scutatus scutatus. FEBS Lett. 1990 Feb 26;261(2):226–230. doi: 10.1016/0014-5793(90)80559-2. [DOI] [PubMed] [Google Scholar]
  7. Cull-Candy S. G., Fohlman J., Gustavsson D., Lüllmann-Rauch R., Thesleff S. The effects of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction. Neuroscience. 1976 Jun;1(3):175–180. doi: 10.1016/0306-4522(76)90074-9. [DOI] [PubMed] [Google Scholar]
  8. Dufton M. J., Eaker D., Hider R. C. Conformational properties of phospholipases A2. Secondary-structure prediction, circular dichroism and relative interface hydrophobicity. Eur J Biochem. 1983 Dec 15;137(3):537–544. doi: 10.1111/j.1432-1033.1983.tb07859.x. [DOI] [PubMed] [Google Scholar]
  9. Halpert J., Eaker D. Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake). J Biol Chem. 1975 Sep 10;250(17):6990–6997. [PubMed] [Google Scholar]
  10. Halpert J., Eaker D. Isolation of a non-neurotoxic, non-enzymatic phospholipase A homologue from the venom of the australian tiger snake Notechis scutatus scutatus. FEBS Lett. 1976 Nov 15;72(1):91–95. doi: 10.1016/0014-5793(76)80905-2. [DOI] [PubMed] [Google Scholar]
  11. Halpert J., Eaker D., Karlsson E. The role of phospholipase activity in the action of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake). FEBS Lett. 1976 Jan 1;61(1):72–76. doi: 10.1016/0014-5793(76)80174-3. [DOI] [PubMed] [Google Scholar]
  12. Halpert J. Structure and function of neuro- and myotoxic phospholipases: modification with ethoxyformic anhydride of notechis II-5 from the venom of the Australian tiger snake Notechis scutatus scutatus. Adv Cytopharmacol. 1979;3:45–62. [PubMed] [Google Scholar]
  13. Harris J. B., Karlsson E., Thesleff S. Effects of an isolated toxin from Australian tiger snake (Notechis scutatus scutatus) venom at the mammalian neuromuscular junction. Br J Pharmacol. 1973 Jan;47(1):141–146. doi: 10.1111/j.1476-5381.1973.tb08168.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harris J. B. Phospholipases in snake venoms and their effects on nerve and muscle. Pharmacol Ther. 1985;31(1-2):79–102. doi: 10.1016/0163-7258(85)90038-5. [DOI] [PubMed] [Google Scholar]
  15. Howard B. D., Gundersen C. B., Jr Effects and mechanisms of polypeptide neurotoxins that act presynaptically. Annu Rev Pharmacol Toxicol. 1980;20:307–336. doi: 10.1146/annurev.pa.20.040180.001515. [DOI] [PubMed] [Google Scholar]
  16. Howard B. D., Truog R. Relationship between the neurotoxicity and phospholipase A activity of beta-bungarotoxin. Biochemistry. 1977 Jan 11;16(1):122–125. doi: 10.1021/bi00620a020. [DOI] [PubMed] [Google Scholar]
  17. Kini R. M., Evans H. J. A model to explain the pharmacological effects of snake venom phospholipases A2. Toxicon. 1989;27(6):613–635. doi: 10.1016/0041-0101(89)90013-5. [DOI] [PubMed] [Google Scholar]
  18. Mollier P., Chwetzoff S., Bouet F., Harvey A. L., Ménez A. Tryptophan 110, a residue involved in the toxic activity but not in the enzymatic activity of notexin. Eur J Biochem. 1989 Nov 6;185(2):263–270. doi: 10.1111/j.1432-1033.1989.tb15111.x. [DOI] [PubMed] [Google Scholar]
  19. Ng R. H., Howard B. D. Deenergization of nerve terminals by beta-bungarotoxin. Biochemistry. 1978 Nov 14;17(23):4978–4986. doi: 10.1021/bi00616a019. [DOI] [PubMed] [Google Scholar]
  20. Ng R. H., Howard B. D. Mitochondria and sarcoplasmic reticulum as model targets for neurotoxic and myotoxic phospholipases A2. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1346–1350. doi: 10.1073/pnas.77.3.1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nolan O., O'Kennedy R. Bifunctional antibodies: concept, production and applications. Biochim Biophys Acta. 1990 Aug 1;1040(1):1–11. doi: 10.1016/0167-4838(90)90139-7. [DOI] [PubMed] [Google Scholar]
  22. Oberfelder R. W., Lee J. C. Measurement of ligand-protein interaction by electrophoretic and spectroscopic techniques. Methods Enzymol. 1985;117:381–399. doi: 10.1016/s0076-6879(85)17023-0. [DOI] [PubMed] [Google Scholar]
  23. Rosenberg P., Ghassemi A., Condrea E., Dhillon D., Yang C. C. Do chemical modifications dissociate between the enzymatic and pharmacological activities of beta bungarotoxin and notexin? Toxicon. 1989;27(2):137–159. doi: 10.1016/0041-0101(89)90128-1. [DOI] [PubMed] [Google Scholar]
  24. Scott D. L., White S. P., Otwinowski Z., Yuan W., Gelb M. H., Sigler P. B. Interfacial catalysis: the mechanism of phospholipase A2. Science. 1990 Dec 14;250(4987):1541–1546. doi: 10.1126/science.2274785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Strong P. N., Goerke J., Oberg S. G., Kelly R. B. beta-Bungarotoxin, a pre-synaptic toxin with enzymatic activity. Proc Natl Acad Sci U S A. 1976 Jan;73(1):178–182. doi: 10.1073/pnas.73.1.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Su M. J., Chang C. C. Presynaptic effects of snake venom toxins which have phospholipase A2 activity (beta-bungarotoxin, taipoxin, crotoxin). Toxicon. 1984;22(4):631–640. doi: 10.1016/0041-0101(84)90003-5. [DOI] [PubMed] [Google Scholar]
  27. Tsai I. H., Liu H. C., Chang T. Toxicity domain in presynaptically toxic phospholipase A2 of snake venom. Biochim Biophys Acta. 1987 Nov 5;916(1):94–99. doi: 10.1016/0167-4838(87)90215-9. [DOI] [PubMed] [Google Scholar]
  28. Tzeng M. C., Hseu M. J., Yen C. H. Taipoxin-binding protein on synaptic membranes: identification by affinity labeling. Biochem Biophys Res Commun. 1989 Dec 15;165(2):689–694. doi: 10.1016/s0006-291x(89)80021-x. [DOI] [PubMed] [Google Scholar]
  29. Ward L. D. Measurement of ligand binding to proteins by fluorescence spectroscopy. Methods Enzymol. 1985;117:400–414. doi: 10.1016/s0076-6879(85)17024-2. [DOI] [PubMed] [Google Scholar]
  30. White S. P., Scott D. L., Otwinowski Z., Gelb M. H., Sigler P. B. Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue. Science. 1990 Dec 14;250(4987):1560–1563. doi: 10.1126/science.2274787. [DOI] [PubMed] [Google Scholar]
  31. Yang C. C., Chang L. S. Role of the N-terminal region in phospholipases A2 from Naja naja atra (Taiwan cobra) and Naja nigricollis (spitting cobra) venoms. Toxicon. 1988;26(8):721–731. doi: 10.1016/0041-0101(88)90279-6. [DOI] [PubMed] [Google Scholar]
  32. Yang C. C., Chang L. S. Studies on the status of lysine residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom. Biochem J. 1989 Sep 15;262(3):855–860. doi: 10.1042/bj2620855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yang C. C., Yang H. J., Chiu R. H. The position of disulfide bonds in cobrotoxin. Biochim Biophys Acta. 1970 Aug 21;214(2):355–363. doi: 10.1016/0005-2795(70)90013-9. [DOI] [PubMed] [Google Scholar]

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