Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Sep 1;278(Pt 2):481–486. doi: 10.1042/bj2780481

Role of the N-terminal region of phospholipase A2 subunit of beta 1-bungarotoxin in the toxin-Ca2+ complex-formation.

S T Chu 1, Y H Chen 1
PMCID: PMC1151370  PMID: 1898340

Abstract

beta 1-Bungarotoxin consists of a phospholipase A2 subunit and a non-phospholipase A2 subunit. Modification of beta 1-bungarotoxin with CNBr resulted in cleavage at Met-6 and Met-8 of its phospholipase A2 subunit. Analysis of the fluorescence data of both the toxin-Ca2+ complex at 300-350 nm and the toxin-Tb3+ complex at 450-650 nm showed the existence of two binding sites for both metal ions on the different domains of the toxin molecule. At pH 7.6 the association constants for the high-affinity and low-affinity sites of the toxin-Ca2+ complex were determined to be 2.79 x 10(3) +/- 0.21 x 10(3) M-1 and 0.47 x 10(3) +/- 0.06 x 10(3) M-1 respectively. For the toxin-Tb3+ complex the association constant for the high-affinity site was 2.95 x 10(3) +/- 0.43 x 10(3) M-1 and that for the low-affinity site was 0.11 x 10(3) +/- 0.03 x 10(3) M-1. Removal of the N-terminal octapeptide of the phospholipase A2 subunit from the toxin molecule caused disintegration of the low-affinity site but did not disrupt the high-affinity site. This might accompany a change in the configuration around His-48 of the phospholipase A2 subunit. Between pH 6 and 8 the binding of metal ions to the high-affinity site increased but that to the low-affinity site did not change with increasing pH. The neurotoxicity and enzymic activity of the toxin were lost on removal of the low-affinity site.

Full text

PDF
481

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abe T., Alemá S., Miledi R. Isolation and characterization of presynaptically acting neurotoxins from the venom of Bungarus snakes. Eur J Biochem. 1977 Oct 17;80(1):1–12. doi: 10.1111/j.1432-1033.1977.tb11849.x. [DOI] [PubMed] [Google Scholar]
  2. Achari A., Scott D., Barlow P., Vidal J. C., Otwinowski Z., Brunie S., Sigler P. B. Facing up to membranes: structure/function relationships in phospholipases. Cold Spring Harb Symp Quant Biol. 1987;52:441–452. doi: 10.1101/sqb.1987.052.01.051. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Chen Y. H., Tai J. C., Huang W. J., Lai M. Z., Hung M. C., Lai M. D., Yang J. T. Role of aromatic residues in the structure-function relationship of alpha-bungarotoxin. Biochemistry. 1982 May 25;21(11):2592–2600. doi: 10.1021/bi00540a003. [DOI] [PubMed] [Google Scholar]
  5. Chen Y. H., Yang J. T., Chau K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. doi: 10.1021/bi00713a027. [DOI] [PubMed] [Google Scholar]
  6. Chen Y. H., Yang J. T., Martinez H. M. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry. 1972 Oct 24;11(22):4120–4131. doi: 10.1021/bi00772a015. [DOI] [PubMed] [Google Scholar]
  7. Chu S. T., Chen Y. H. The intrinsic tryptophan fluorescence of beta 1-bungarotoxin and the Ca2+-binding domains of the toxin as probed with Tb3+ luminescence. Biochem J. 1989 Sep 15;262(3):773–779. doi: 10.1042/bj2620773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dijkstra B. W., Drenth J., Kalk K. H., Vandermaelen P. J. Three-dimensional structure and disulfide bond connections in bovine pancreatic phospholipase A2. J Mol Biol. 1978 Sep 5;124(1):53–60. doi: 10.1016/0022-2836(78)90146-8. [DOI] [PubMed] [Google Scholar]
  9. Dijkstra B. W., Kalk K. H., Drenth J., de Haas G. H., Egmond M. R., Slotboom A. J. Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2. Biochemistry. 1984 Jun 5;23(12):2759–2766. doi: 10.1021/bi00307a035. [DOI] [PubMed] [Google Scholar]
  10. Dijkstra B. W., Kalk K. H., Hol W. G., Drenth J. Structure of bovine pancreatic phospholipase A2 at 1.7A resolution. J Mol Biol. 1981 Mar 25;147(1):97–123. doi: 10.1016/0022-2836(81)90081-4. [DOI] [PubMed] [Google Scholar]
  11. Dijkstra B. W., Renetseder R., Kalk K. H., Hol W. G., Drenth J. Structure of porcine pancreatic phospholipase A2 at 2.6 A resolution and comparison with bovine phospholipase A2. J Mol Biol. 1983 Jul 25;168(1):163–179. doi: 10.1016/s0022-2836(83)80328-3. [DOI] [PubMed] [Google Scholar]
  12. Dufton M. J. Proteinase inhibitors and dendrotoxins. Sequence classification, structural prediction and structure/activity. Eur J Biochem. 1985 Dec 16;153(3):647–654. doi: 10.1111/j.1432-1033.1985.tb09349.x. [DOI] [PubMed] [Google Scholar]
  13. Epstein M., Levitzki A., Reuben J. Binding of lanthanides and of divalent metal ions to porcine trypsin. Biochemistry. 1974 Apr 9;13(8):1777–1782. doi: 10.1021/bi00705a034. [DOI] [PubMed] [Google Scholar]
  14. Fisher J., Primrose W. U., Roberts G. C., Dekker N., Boelens R., Kaptein R., Slotboom A. J. 1H NMR studies of bovine and porcine phospholipase A2: assignment of aromatic resonances and evidence for a conformational equilibrium in solution. Biochemistry. 1989 Jul 11;28(14):5939–5946. doi: 10.1021/bi00440a034. [DOI] [PubMed] [Google Scholar]
  15. GROSS E., WITKOP B. Nonenzymatic cleavage of peptide bonds: the methionine residues in bovine pancreatic ribonuclease. J Biol Chem. 1962 Jun;237:1856–1860. [PubMed] [Google Scholar]
  16. Harvey A. L., Karlsson E. Protease inhibitor homologues from mamba venoms: facilitation of acetylcholine release and interactions with prejunctional blocking toxins. Br J Pharmacol. 1982 Sep;77(1):153–161. doi: 10.1111/j.1476-5381.1982.tb09281.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keith C., Feldman D. S., Deganello S., Glick J., Ward K. B., Jones E. O., Sigler P. B. The 2.5 A crystal structure of a dimeric phospholipase A2 from the venom of Crotalus atrox. J Biol Chem. 1981 Aug 25;256(16):8602–8607. [PubMed] [Google Scholar]
  18. Kelly R. B., Oberg S. G., Strong P. N., Wagner G. M. beta-Bungarotoxin, a phospholipase that stimulates transmitter release. Cold Spring Harb Symp Quant Biol. 1976;40:117–125. doi: 10.1101/sqb.1976.040.01.013. [DOI] [PubMed] [Google Scholar]
  19. Kini R. M., Evans H. J. Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2. J Biol Chem. 1987 Oct 25;262(30):14402–14407. [PubMed] [Google Scholar]
  20. Kondo K., Narita K., Lee C. Y. Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait). J Biochem. 1978 Jan;83(1):91–99. doi: 10.1093/oxfordjournals.jbchem.a131917. [DOI] [PubMed] [Google Scholar]
  21. Lin W. Z., Chu S. T., Chen Y. H. Optical activity and conformation of beta-bungarotoxin in solution. Proc Natl Sci Counc Repub China B. 1984 Apr;8(2):113–118. [PubMed] [Google Scholar]
  22. Renetseder R., Brunie S., Dijkstra B. W., Drenth J., Sigler P. B. A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atrox venom. J Biol Chem. 1985 Sep 25;260(21):11627–11634. [PubMed] [Google Scholar]
  23. Wang C. L., Leavis P. C., Horrocks W. D., Jr, Gergely J. Binding of lanthanide ions to troponin C. Biochemistry. 1981 Apr 28;20(9):2439–2444. doi: 10.1021/bi00512a012. [DOI] [PubMed] [Google Scholar]
  24. van Scharrenburg G. J., Puijk W. C., Egmond M. R., van der Schaft P. H., de Haas G. H., Slotboom A. J. Effects of substitution of the absolutely invariant glutamine-4 and phenylalanine-5 in bovine pancreatic phospholipase A2 on enzymatic activity and substrate binding properties. Biochemistry. 1982 Mar 16;21(6):1345–1352. doi: 10.1021/bi00535a037. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES