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. 1995 May 15;308(Pt 1):297–304. doi: 10.1042/bj3080297

Chemical modification and inactivation of phospholipases A2 by a manoalide analogue.

S Fujii 1, Y Tahara 1, M Toyomoto 1, S Hada 1, H Nishimura 1, S Inoue 1, K Ikeda 1, Y Inagaki 1, S Katsumura 1, Y Samejima 1, et al.
PMCID: PMC1136876  PMID: 7755577

Abstract

Chemical modification and inactivation of bovine pancreatic, porcine pancreatic, Naja naja atra and Pseudechis australis phospholipases A2 (PLA2s), belonging to Group I, and of Trimeresurus flavoviridis, Vipera russelli russelli and Agkistrodon halys blomhoffii PLA2s, belonging to Group II, were investigated by the use of a manoalide (MLD)-analogue, 1-(2,5-dihydro-hydroxy-5-oxo-3-furanyl)-8,12-dimethyl-4-formyl-3,7, 11-tridecatrienol. At appropriate time intervals, residual PLA2 activities towards monodispersed, anionic mixed micellar and non-ionic mixed micellar substrates were measured. We tested the protective effect of micellar n-dodecylphosphocholine (n-C12PC) on enzyme inactivation. Inactivation of pancreatic PLA2s (Group I) was only observed towards anionic mixed micellar substrates. This inactivation was completely prevented by the presence of micellar n-C12PC. From a fragmentation study of modified bovine pancreatic PLA2 using lysyl endopeptidase, we speculated that Lys-56 of this enzyme was modified by MLD-analogue and that this modification was responsible for enzyme inactivation. Inactivation of non-pancreatic PLA2s was observed towards all types of substrate, except that no significant inactivation of N. naja atra PLA2 (Group I) towards monodispersed substrate was noted. Micellar n-C12PC protected N. naja atra PLA2 (Group I) completely from inactivation by MLD-analogue, but had lesser protective effects on P. australis PLA2 (Group I), T. flavoviridis and V. russelli russelli PLA2s (Group II). However, no significant protection of A. halys blomhoffii PLA2s (Group II) activity was observed. These results indicate that the inactivation of pancreatic and N. naja atra PLA2s originates from the modification of Lys residues at the interfacial recognition site, and that inactivation of P. australis, T. flavoviridis and V. russelli PLA2s arises from the modification of Lys residues at the catalytic site, interfacial recognition site and regions outside both sites. The inactivation of A. halys blomhoffii PLA2 was assumed to be due to the modification of Lys residues outside the two sites described above.

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Selected References

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  1. Bennett C. F., Mong S., Clarke M. A., Kruse L. I., Crooke S. T. Differential effects of manoalide on secreted and intracellular phospholipases. Biochem Pharmacol. 1987 Mar 1;36(5):733–740. doi: 10.1016/0006-2952(87)90727-1. [DOI] [PubMed] [Google Scholar]
  2. Cavins J. F., Friedman M. An internal standard for amino acid analyses: S-beta-(4-pyridylethyl)-L-cysteine. Anal Biochem. 1970 Jun;35(2):489–493. doi: 10.1016/0003-2697(70)90211-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Donné-Op den Kelder G. M., de Haas G. H., Egmond M. R. Localization of the second calcium ion binding site in porcine and equine phospholipase A2. Biochemistry. 1983 May 10;22(10):2470–2478. doi: 10.1021/bi00279a025. [DOI] [PubMed] [Google Scholar]
  6. Glaser K. B., Jacobs R. S. Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2. Biochem Pharmacol. 1986 Feb 1;35(3):449–453. doi: 10.1016/0006-2952(86)90218-2. [DOI] [PubMed] [Google Scholar]
  7. Glaser K. B., Vedvick T. S., Jacobs R. S. Inactivation of phospholipase A2 by manoalide. Localization of the manoalide binding site on bee venom phospholipase A2. Biochem Pharmacol. 1988 Oct 1;37(19):3639–3646. doi: 10.1016/0006-2952(88)90396-6. [DOI] [PubMed] [Google Scholar]
  8. Heinrikson R. L., Krueger E. T., Keim P. S. Amino acid sequence of phospholipase A2-alpha from the venom of Crotalus adamanteus. A new classification of phospholipases A2 based upon structural determinants. J Biol Chem. 1977 Jul 25;252(14):4913–4921. [PubMed] [Google Scholar]
  9. Kawauchi S., Iwanaga S., Samejima Y., Suzuki T. Isolation and characterization of two phospholipase A's from the venom of Agkistrodon lays blomhoffii. Biochim Biophys Acta. 1971 Apr 27;236(1):142–160. doi: 10.1016/0005-2795(71)90159-0. [DOI] [PubMed] [Google Scholar]
  10. Lombardo D., Dennis E. A. Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor. J Biol Chem. 1985 Jun 25;260(12):7234–7240. [PubMed] [Google Scholar]
  11. Miyake T., Inoue S., Ikeda K., Teshima K., Samejima Y., Omori-Satoh T. pH dependence of the reaction rate of His 48 with p-bromophenacyl bromide and of the binding constant to Ca2+ of the monomeric forms of intact and alpha-NH2 modified phospholipases A2 from Trimeresurus flavoviridis. J Biochem. 1989 Apr;105(4):565–572. doi: 10.1093/oxfordjournals.jbchem.a122705. [DOI] [PubMed] [Google Scholar]
  12. Nieuwenhuizen W., Kunze H., de Haas G. H. Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from porcine pancreas. Methods Enzymol. 1974;32:147–154. doi: 10.1016/0076-6879(74)32018-6. [DOI] [PubMed] [Google Scholar]
  13. Noel J. P., Bingman C. A., Deng T. L., Dupureur C. M., Hamilton K. J., Jiang R. T., Kwak J. G., Sekharudu C., Sundaralingam M., Tsai M. D. Phospholipase A2 engineering. X-ray structural and functional evidence for the interaction of lysine-56 with substrates. Biochemistry. 1991 Dec 24;30(51):11801–11811. doi: 10.1021/bi00115a010. [DOI] [PubMed] [Google Scholar]
  14. Scott D. L., White S. P., Browning J. L., Rosa J. J., Gelb M. H., Sigler P. B. Structures of free and inhibited human secretory phospholipase A2 from inflammatory exudate. Science. 1991 Nov 15;254(5034):1007–1010. doi: 10.1126/science.1948070. [DOI] [PubMed] [Google Scholar]
  15. Takasaki C., Suzuki J., Tamiya N. Purification and properties of several phospholipases A2 from the venom of Australian king brown snake (Pseudechis australis). Toxicon. 1990;28(3):319–327. doi: 10.1016/0041-0101(90)90067-h. [DOI] [PubMed] [Google Scholar]
  16. Takasaki C., Yutani F., Kajiyashiki T. Amino acid sequences of eight phospholipases A2 from the venom of Australian king brown snake, Pseudechis australis. Toxicon. 1990;28(3):329–339. doi: 10.1016/0041-0101(90)90068-i. [DOI] [PubMed] [Google Scholar]
  17. Tanaka S., Mohri N., Kihara H., Ohno M. Amino acid sequence of Trimeresurus flavoviridis phospholipase A2. J Biochem. 1986 Jan;99(1):281–289. doi: 10.1093/oxfordjournals.jbchem.a135471. [DOI] [PubMed] [Google Scholar]
  18. Teshima K., Ikeda K., Hamaguchi K., Hayashi K. Bindings of cobra venom phospholipases A2 to micelles of n-hexadecylphosphorylcholine. J Biochem. 1983 Jul;94(1):223–232. doi: 10.1093/oxfordjournals.jbchem.a134333. [DOI] [PubMed] [Google Scholar]
  19. Teshima K., Ikeda K., Hamaguchi K., Hayashi K. Bindings of monodispersed n-alkylphosphorylcholines to cobra venom phospholipases A2. J Biochem. 1981 Apr;89(4):1163–1174. [PubMed] [Google Scholar]
  20. Teshima K., Ikeda K., Hamaguchi K., Hayashi K. pH dependence of the binding constant of Ca2+ to cobra venom phospholipases A2. J Biochem. 1981 Jan;89(1):13–20. doi: 10.1093/oxfordjournals.jbchem.a133173. [DOI] [PubMed] [Google Scholar]
  21. Teshima K., Ikeda K., Miyake T., Imamura M., Inoue S., Samejima Y., Kawauchi S., Omori-Satoh T. Interactions of monodispersed and micellar substrates with a phospholipase A2 from Trimeresurus flavoviridis. J Biochem. 1989 Jun;105(6):1044–1051. doi: 10.1093/oxfordjournals.jbchem.a122764. [DOI] [PubMed] [Google Scholar]
  22. Thunnissen M. M., Ab E., Kalk K. H., Drenth J., Dijkstra B. W., Kuipers O. P., Dijkman R., de Haas G. H., Verheij H. M. X-ray structure of phospholipase A2 complexed with a substrate-derived inhibitor. Nature. 1990 Oct 18;347(6294):689–691. doi: 10.1038/347689a0. [DOI] [PubMed] [Google Scholar]
  23. Tomoo K., Ohishi H., Doi M., Ishida T., Inoue M., Ikeda K., Mizuno H. Interaction mode of n-dodecylphosphorylcholine, a substrate analogue, with bovine pancreas phospholipase A2 as determined by X-ray crystal analysis. Biochem Biophys Res Commun. 1992 Sep 16;187(2):821–827. doi: 10.1016/0006-291x(92)91270-z. [DOI] [PubMed] [Google Scholar]
  24. Tomoo K., Ohishi H., Ishida T., Inoue M., Ikeda K., Aoki Y., Samejima Y. Revised amino acid sequence, crystallization, and preliminary x-ray diffraction analysis of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii. J Biol Chem. 1989 Feb 25;264(6):3636–3638. [PubMed] [Google Scholar]
  25. Verger R. Interfacial enzyme kinetics of lipolysis. Annu Rev Biophys Bioeng. 1976;5:77–117. doi: 10.1146/annurev.bb.05.060176.000453. [DOI] [PubMed] [Google Scholar]
  26. Volwerk J. J., Jost P. C., de Haas G. H., Griffith O. H. Activation of porcine pancreatic phospholipase A2 by the presence of negative charges at the lipid-water interface. Biochemistry. 1986 Apr 8;25(7):1726–1733. doi: 10.1021/bi00355a042. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. de Araujo P. S., Rosseneu M. Y., Kremer J. M., van Zoelen E. J., de Haas G. H. Structure and thermodynamic properties of the complexes between phospholipase A2 and lipid micelles. Biochemistry. 1979 Feb 20;18(4):580–586. doi: 10.1021/bi00571a005. [DOI] [PubMed] [Google Scholar]
  29. de Haas G. H., Postema N. M., Nieuwenhuizen W., van Deenen L. L. Purification and properties of an anionic zymogen of phospholipase A from porcine pancreas. Biochim Biophys Acta. 1968 Apr 24;159(1):118–129. doi: 10.1016/0005-2744(68)90249-0. [DOI] [PubMed] [Google Scholar]

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