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. 1989 Sep 15;262(3):855–860. doi: 10.1042/bj2620855

Studies on the status of lysine residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.

C C Yang 1, L S Chang 1
PMCID: PMC1133352  PMID: 2511834

Abstract

Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulphonic acid (TNBS), and two major trinitrophenylated (TNP) derivatives, TNP-1 and TNP-2, were separated by h.p.l.c. TNP-1 contained only one TNP group on Lys-6 and showed a marked decrease in enzymic activity, but still retained 45% of the lethal toxicity. Both Lys-6 and Lys-65 were modified in TNP-2, and modification of Lys-65 caused a further reduction of the lethal toxicity to 12.6%. However, the antigenicity of both TNP-1 and TNP-2 remained unchanged. The reactivity of Lys-6 and Lys-65 toward TNBS was greatly enhanced by Ca2+ and dihexanoyl-lecithin, suggesting that the two Lys residues are not directly involved in the binding of Ca2+ and substrate. The modified derivatives retained their affinity for Ca2+, indicating that Lys-6 and Lys-65 did not participate in the Ca2+ binding. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated PLA2 are almost the same as those of native PLA2. These results indicate that Lys-6 and Lys-65 are important for the biological activities of PLA2, and incorporation of a bulky TNP group on Lys-6 and Lys-65 might give rise to a distortion of the active conformation of PLA2.

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Selected References

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  1. 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]
  2. Barrington P. L., Yang C. C., Rosenberg P. Cardiotoxic effects of Naja nigricollis venom phospholipase A2 are not due to phospholipid hydrolytic products. Life Sci. 1984 Aug 27;35(9):987–995. doi: 10.1016/0024-3205(84)90665-9. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. CRESTFIELD A. M., MOORE S., STEIN W. H. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem. 1963 Feb;238:622–627. [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. Condrea E., Fletcher J. E., Rapuano B. E., Yang C. C., Rosenberg P. Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipases A2. Toxicon. 1981;19(5):705–720. doi: 10.1016/0041-0101(81)90108-2. [DOI] [PubMed] [Google Scholar]
  7. Condrea E., Fletcher J. E., Rapuano B. E., Yang C. C., Rosenberg P. Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipases A2 from Hemachatus haemachatus and Naja atra snake venoms. Toxicon. 1981;19(1):61–71. doi: 10.1016/0041-0101(81)90118-5. [DOI] [PubMed] [Google Scholar]
  8. Condrea E., Yang C. C., Rosenberg P. Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom--I. Enzymatic activity on free and membrane bound substrates. Biochem Pharmacol. 1980 Jun 1;29(11):1555–1563. doi: 10.1016/0006-2952(80)90608-5. [DOI] [PubMed] [Google Scholar]
  9. Condrea E., Yang C. C., Rosenberg P. Lack of correlation between anticoagulant activity and phospholipid hydrolysis by snake venom phospholipases A2. Thromb Haemost. 1981 Feb 23;45(1):82–85. [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. Fletcher J. E., Rapuano B. E., Condrea E., Yang C. C., Rosenberg P. Relationship between catalysis and toxicological properties of three phospholipases A2 from elapid snake venoms. Toxicol Appl Pharmacol. 1981 Jun 30;59(2):375–388. doi: 10.1016/0041-008x(81)90209-x. [DOI] [PubMed] [Google Scholar]
  13. Fletcher J. E., Rapuano B. E., Condrea E., Yang C. C., Ryan M., Rosenberg P. Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom--II. Pharmacological properties in relationship to enzymatic activity. Biochem Pharmacol. 1980 Jun 1;29(11):1565–1574. doi: 10.1016/0006-2952(80)90609-7. [DOI] [PubMed] [Google Scholar]
  14. Fletcher J. E., Yang C. C., Rosenberg P. Basic phospholipase A2 from Naja nigricollis snake venom: phospholipid hydrolysis and effects on electrical and contractile activity of the rat heart. Toxicol Appl Pharmacol. 1982 Oct;66(1):39–54. doi: 10.1016/0041-008x(82)90059-x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kini R. M., Iwanaga S. Structure-function relationships of phospholipases. I: Prediction of presynaptic neurotoxicity. Toxicon. 1986;24(6):527–541. doi: 10.1016/0041-0101(86)90173-x. [DOI] [PubMed] [Google Scholar]
  17. Maraganore J. M., Heinrikson R. L. The role of lysyl residues of phospholipases A2 in the formation of the catalytic complex. Biochem Biophys Res Commun. 1985 Aug 30;131(1):129–138. doi: 10.1016/0006-291x(85)91780-2. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Rosenberg P., Condrea E., Rapuano B. E., Soons K. R., Yang C. C. Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A2 by modification of carboxylate groups. Biochem Pharmacol. 1983 Dec 1;32(23):3525–3530. doi: 10.1016/0006-2952(83)90298-8. [DOI] [PubMed] [Google Scholar]
  21. Soons K. R., Condrea E., Yang C. C., Rosenberg P. Effects of modification of tyrosines 3 and 62 (63) on enzymatic and toxicological properties of phospholipases A2 from Naja nigricollis and Naja naja atra snake venoms. Toxicon. 1986;24(7):679–693. doi: 10.1016/0041-0101(86)90031-0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Yang C. C., King K., Sun T. P. Chemical modification of lysine and histidine residues in phospholipase A2 from the venom of Naja naja atra (Taiwan cobra). Toxicon. 1981;19(5):645–659. doi: 10.1016/0041-0101(81)90102-1. [DOI] [PubMed] [Google Scholar]
  25. Zan Y. P., Condrea E., Yang C. C., Rosenberg P. Phospholipid hydrolysis in serum lipoproteins by a basic phospholipase A2 from Naja nigricollis snake venom and an acidic phospholipase A2 from Naja naja atra snake venom. Toxicon. 1983;21(4):481–490. doi: 10.1016/0041-0101(83)90126-5. [DOI] [PubMed] [Google Scholar]

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