미국자리공(Phytolacca americana L.) 뿌리의 항균 펩타이드 정제 및 특성연구

Purification and Charaterization of Antimicrobial Peptide from Roots of Pokeweed

  • 발행 : 2003.11.30

초록

미국자리공(Phytolacca americana L.) 뿌리에서 항균 펩타이드를 분리하여 PAMP-r로 명명 하였다. 분리는 DEAE-cellulose, sephadex G-75, Mono S, Resource RPC등을 거쳐 이루어 졌으며 최종산물은 15% SDS-PAGE 에서 약 4,900 Da의 분자량을 보였다. PAMP-r는 광범위한 항균활성을 나타냈으며 온도와 pH변화에도 높은 안정성을 보였다. 항균활성은 $80^{\circ}C$에서 30분동안 유지될 뿐만 아니라 pH도 3.0의 산성에서부터 pH 8.0의 알카리에 이르기까지 광범위한 범위 안에서 안정하게 나타났다.

An antimicrobial peptide was purified from the roots of Phytolacca americana L. and was designated as PAMP-r. Purification was carried out by DEAE-cellulose anion exchange, sephadex G-75 gel filtration, Mono S cation exchange, and Resource RPC reverse phage chromatography. The molecular weight of PAMP-r was estimated to be about 4,900 Da by 15% SDS-PAGE under reducing condition. PAMP-r exhibited a broad spectrum of antimicrobial activity. PAMP-r was stable against heat and pH treatment; its activity was not diminished by the heat treatment up to $80^{\circ}C$ for 30 min, and it showed a pH stability in the range between pH 3.0 to pH 8.0.

키워드

참고문헌

  1. Agrios, G. N. (1988) In Plant Pathology. Academic Press. Inc. New York, pp. 325-450
  2. Bowles, D. J. (1990) Defense-related proteins in higher plants. Annu. Rev. Biochem. 59, 873-907 https://doi.org/10.1146/annurev.bi.59.070190.004301
  3. Schlumbaum, A., Mauch, F., Vogeli U. and Boller T. (1986) Plant chitinases are potent inhibitors of fungal growth. Nature 324, 365-367 https://doi.org/10.1038/324365a0
  4. Kim, W. Y., Cheong, N. E., Jae, D. Y., Lee, D. C., Kim, J. W., Cho S. H. and Lee, S. Y. (1994) Characterization of antimicrobial chitinase purified from the grapefruit extract. Korean J. Plant Pathol. 10, 277-283
  5. Arlorio, M., Ludwig, A., Boller T. and Bonfante P. (1992) Inhibition of fungal growth by plant chitinases and $\beta$-l, 3-glucanases a morphological study. Protoplasma 171, 34-43 https://doi.org/10.1007/BF01379278
  6. Stirpe, F. and Barbieri, L. (1986) Ribosome-inactivating proteins up to date. FEBS Lett. 195, 1-8 https://doi.org/10.1016/0014-5793(86)80118-1
  7. lrvin, J. D. and Uckun, F. M. (1992) Pokeweed antiviral protein: Ribosome inactivation and therapeutic applications. Pharmaco. Therapeut. 55, 279-302 https://doi.org/10.1016/0163-7258(92)90053-3
  8. Tumer, N. E., Hudak, K., Di, R., Coetzer, C., Wang P. and Zourenko O. (1999) Pokeweed antiviral protein and its applications. Curr. Top. Microbiol. Immunol. 240, 139-158
  9. Bohlmann, H., Apel, K. and Garcia-Olmedo F. (1994) Thionins. Plant Mol. Biol. Rep. 12, S75 https://doi.org/10.1007/BF02671578
  10. Broekaert, W. F., Cammue, B. P. A., De Bolle M. F. C., Thevissen, K., De Samblanx G. W. and Osbom R. W. (1997) Antimicrobial peptides from plants. Crit. Rev. Plant Sci. 16, 297-323 https://doi.org/10.1080/713608148
  11. Smith, G. R, Patel, S. U., Windass, J. D., Thornton, J. M., Winter G. and Griffiths, A. D. (1998) Small binding proteins selected from a combinatorial repertoire of knottins displayed on phage. J. Mol. Biol. 277, 317-332 https://doi.org/10.1006/jmbi.1997.1621
  12. Irvin, J. D. (1975) Purification and partial characterization of the antiviral protein from Phytolacca americana which inhibits eukaryotic protein synthesis. Arch. Biochem. Biophys. 169, 522-528 https://doi.org/10.1016/0003-9861(75)90195-2
  13. Myers D. E., Jun, X., Clementson, D., Donelson, R., Sicheneder, A., Hoffman, N., Bell, K., Sarquis, M., Langlie, M. C., Turner N. and Uckun, F. M. (1997) Large scale manufacturing of TXU (anti-CD7)-pokeweed antiviral protein (PAP) immunoconjugate for clinical trials. Leuk. Lymphoma 27, 275-302
  14. Kino M., Yamaguchi, K., Umekawa, H. and Funatsu, G. (1995) Purification and characterization of three mitogenic lectins from the roots of pokeweed (Phytolacca americana). Biosci. Biotech. Biochem. 59, 683-688 https://doi.org/10.1271/bbb.59.683
  15. Shao, F., Hu, Z., Xiong, Y. M., Huang, Q. Z., Wang, C. G., Zhu R. H. and Wang D. C. (1999) A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning. BBA. 1430, 262-268 https://doi.org/10.1016/S0167-4838(99)00013-8
  16. Liu, Y., Luo, J., Xu, C., Ren, F., Peng, C., Wu, G. and Zhao, J. (2000) Purification, characterization, and molecular cloning of the gene of a seed-spectfic antimicrobial protein from pokeweed. Plant Physiol. 122, 1015-1024 https://doi.org/10.1104/pp.122.4.1015
  17. Son, D. Y., Shin, B. C., Yun, D. J., Seong K. Y., and Chung, Y. R. (1998) Purification and characterization of an antifungal peptide from the seeds of Phytolacca americana. Korean J. Plant Pathol. 14, 203-208
  18. Kim, J. H. (1998) Purification and characterization of lectins from a pokeweed (phytolacca Americana). Kyungsung Univ. Bull. 19, 603-613
  19. Leammli U. K. (1970) Cleavage of stmctural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  20. Hussain, R. F., Nouri A. M. E. and Oliver R. T. D. (1993) A new approach for measurement of cytotoxicity using colorimetric assay. J. Immunol. Methods 160, 89-96 https://doi.org/10.1016/0022-1759(93)90012-V
  21. Sladowski, D., Steer, S. J., Clothier, R. H. and Balls M. (1993) An improved MTT assay. J. Immunol. Methods 157, 203-207 https://doi.org/10.1016/0022-1759(93)90088-O
  22. Mosmann, T., (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63 https://doi.org/10.1016/0022-1759(83)90303-4
  23. Stevens, M. G., Kehrli, M. E. Jr. and Canning, P. C. (1991) A colorimetric assay for quantitating bovine neutrophil bactericidal activity. Vet. Immunol. Immunop. 28, 45-56 https://doi.org/10.1016/0165-2427(91)90042-B
  24. Paik, I. K (1998) Rapid antibacterial sensidvity test by Tetrazolium salt MTT, M.D. Thesis, Chonnam Univ. Jeonju, Korea