겨우살이 Lectin의 정제 및 생화학적 특성

Purification and Biochemical Characterization of Lectin from Viscum album

  • 발행 : 1999.10.01

초록

겨우살이가 가지고 있는 lectin의 약리작용의 가능성에 대한 연구의 일환으로, lectin을 분리 . 정제하였으며, 분자량, subunit수, 탄수화물의 조성, 단백질의 아미노산 조성, 적혈구 응집력, 탄수화물 특이성, pH 및 열 안정성등의 생화학적 특성을 연구하였다. Lectin의 분리는 0.15 M NaCl에 의한 추출, $(NH_4)_2SO_4$ 침전, sepharose 4B affinity chromatography 및 sephadex G-150 gel filtration에 의해 11.64배 정제에 0.14%의 수율을 획득하였으며, HPLC와 전기영동을 이용하여 순도를 검정하였다. Gel filtration에 의해 분석된 lectin의 분자량은 124,000 Da이였고, SDS-PAGE에 의해 분자량이 30,000과 32,000 Da인 2개의 band로 나타나서 각각 2개의 subunit를 갖는 tetramer로 확인되었다. 겨우살이 lectin의 탄수화물은 glucose, fructose, arabinose 및 xylose를 함유하고 있다. 또한 phenlylalanine, lysine, tyrosine, aspartic acid가 비교적 풍부하였고 methionine, alanine, arginine은 비교적 적었다. Lectin은 적혈구에 종 특이성을 나타내는데 겨우살이의 혈구응집반응에서는 사람혈액형간의 차이는 없었으며, 쥐에서 가장 높았고 소에서 가장 낮았으므로 동물 종간에 대한 특이성은 뚜렸하였다. Lectin은 당 특이성을 가지는데, D-galactose, D-mannose, D-lactose 및 D-raffinose에 대해 특이성을 나타내었다. 겨우살이 lectin은 pH4~7에서 안정하였으며, 5$0^{\circ}C$까지는 10~30분 열처리해도 활성이 유지되는 열 안정성 단백질이었다.

The lectin was purified through 0.15 M NaCl extraction, ammonium sulfate precipitation, sepharose 4B affinity chromatography and gel filtration using sephadex G-150 from the leaves of Visum album collected in Mt. Duk Yu. The final gel filtration step resulted in 11.64 folds purification with 0.14% of recovery yield. We also performed biochemical characterization of the purified Visum album lectin. HPLC analysis of lectin purified by gel filtration revealed a singel peak. The analysis of the purified lectin by SDS-PAGE showed a tetramer composed of two identical subunits with molecular weights of 32 and 30 kDa. The lectin was a glycoprotein containing 14.4% carbohydrate, which consist of glucose, fructose, arabinose and xylose, and the amino acids such as phenylalanine, lysine and tyrosine. The purified lectin agglutinated human red blood cell types with similar potency, but when tested against red blood cells from mouse, bovine, rabbit, chicken and porcine, significant difference in potency were observed. Hemaggluting activity was inhibited by D-galactose, D-mannose, D-lactose and D-raffinose, but not by D-glucose, D-glucosamine, D-mannosamine, L-fructose, D-xylose, D-arabinose, D-galacturonic acid, D-fructose, L-rhamnose and N-acetyl-D-galactosamine. The optimal pH and thermal stability of the purified lectin were pH 4.0-7.0 and 20-5$0^{\circ}C$, respectively.

참고문헌

  1. Brit. Med. J. v.15 Hacmagglutinins in seeds Bird, G. W. G.
  2. Chemotaxonomy of the Leguminosae Toms, G. C.;A. Western
  3. Nature v.285 What should be called a lectim Goldstein, I. J.;R. C. Hughes;M. Monsigny;T. Osawa;N. Sharon
  4. J. Bacteriol. v.32 The identification of the hemagglutinin of the jack bean with concanavalin A Sumner, J. B.;S. F. Howell
  5. Proc. Natl. Acad. Sci. U.S.A. v.50 Reactions of normal and tumor cell surfaces to enzymes. I. Wheat germ lipase and associated mucopolysaccharides Aub, J. C.;C. Tieslau;A. Lankester
  6. Ann. Rev. Biochem. v.51 Carbohydrate-specific receptors of the liver Ashwell, G.;J. Harford
  7. Zbl. Bakt. I. Abt. Org. v.45 Beobachtungen uber hamolyse und hamagglutination Landsteiner, K.;H. Raubitschek
  8. Ann. Med. Exp. Biol. Fenn. v.26 Studies on hemagglutinins present in seeds of some representatives of the family of leguminosae Renkonen, K. O.
  9. J. Immunol. v.62 Hemagglutinating substances in various plants Boyd, W. C.;R. M. Reguelra
  10. Science v.177 Lectins: Cell-agglutinating and sugar-specific proteins Sharon, N.;H. Lis.
  11. J. Immunol. v.115 Peanut agglutinin, a new mitogen that binds to galactosyl site exposed after neuraminidase treatment Novogrodsky, A.;R. Lotan;A. Ravid;N. Sharon
  12. Int. Rev. Cytol. v.39 The interaction of lectins with animal cell surfaces Nicolson, G. L.
  13. Lectins. Their chemistry and application to immunology, in the antigens Lis, H.;N. Sharon;Sela M.(ed.)
  14. Biochim. Biophys. Acta v.332 Immunological mechanism of action of the tumor reducing receptor from concanavalin A Gordon, J. A.;M. D. Marquardt
  15. Transplantation v.14 In vivo effects of concanavalin A Nirmul, G.;C. Seeverin;R. N. Taub
  16. Eur. J. Immunol. v.14 Activation of human T lymphocytes: involvement of the T 3 antigen in polyclonal T cel activation by mitogenic lectins and oxidation Fleischer, B.
  17. Nature v.258 Lectins stimulate pollen germination Southworth, D.
  18. Nature v.256 Inhibition of fungal growth by wheat germ agglutinin Mirelman, D.;E. Galun;N. Sharon;R. Lotan
  19. Biochemistry v.20 Evidence that the insulin-like activities of concanavalin A and insulin are mediated by a common insulin receptor linked effector system Howard, M. K.;P. V. Pasquale;A. M. Richard;G. G. Barbara
  20. The biology of mistletoes Lamont, B.;M. Calder(ed.);P. Bernhardt(ed.)
  21. Biochemistry v.13 Mistletoe, myth magic and medicine Griggs, P.
  22. Hort Technology v.2 Mistletoe: Its role in horticulture and human life Paine, L.;H. Harrison
  23. Experientia v.34 The lectin from Viscum album L. purification by bispecific affinity chromatography Ziska, O.;H. Franz;A. Kindt
  24. Bioch. Biophy. Acta v.310 The phytohemagglutinin from sunn hemp seeds(Crotalaria juncea). Purification by biospecific affinity chromatography Ersson, B.;K. Aspberg;J. Porath
  25. Biochem. J. v.91 Estimation of the molecular weights of proteins by sephadex gel filtration Andrews, P.
  26. J. Biol. Chem. v.244 The reiiability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis Weber, K.;M. Osborn
  27. Tappi v.53 A gas chromatographic method for carbohydrates as alditol acetate Bochardt, L. G.;C. V. Piper
  28. Tappi v.65 Improvement of chromatographic method for carbohydrates as alditol acetate Bochardt, L. G.;D. B. Easty
  29. Tappi v.70 Determination of carvohydrates by gas chromatography Vidal, T.;P. J. F. Colom.
  30. J. Biol. Chem. v.251 Complete amino acid analysis of protein's from a single hydrolysate Simpson, R. J.;M. R. Neuberger;T. Y. Lieu
  31. International symposium on immunological method of biological study v.4 The use of a microtitrator in serological procedures Takatsy, G.
  32. Carbohydrate. Research v.50 The isolation of lectins on acid-treated agarose Allen, H. J.;E. A. Johnson
  33. The biology of mistletoes Mistletoes in focus: An introduction Calder, D. M.;M. Calder(ed.);P. Bernhardt(ed.)
  34. Bul. Hist. Med. v.7 Mistletoe, magic and medicine Kanner, L.
  35. Science v.246 Lectins as cell recognition molecule Sharon, N.;H. Lis
  36. J. Biol. Chem. v.243 Protein-carbohydrate interaction. ⅩⅢ. The interaction of concanavalin A with α-mannans from a variety of microorganisms So, L. L.;I. J. Goldstein
  37. Int. J. Biochem. v.11 The lectin from Viscum album L. Isolation, characterization, properties and structure Luther, P.;H. Theise;B. Chatterjee;D. Karduck;G. Uhlenbruck
  38. Plant lectin v.832 The biochemistry of plant lectins (Phytohemagglutinins) Lis, H.;N. Sharon
  39. Biochemistry v.5 Some physical and chemical properties of wheat germ. the phytohemagglutinin of the wheat germ agglutinin Irvin, H.
  40. J. Exp. Med. v.124 Studies on human peripheral blood lymphocytes in vitro I. Biological and physicochemical properties of the pokeweed mitogen Borjeson, J.;R. A. Reisfeld;L. N. Chessin;P. D. Welsh;S. D. Douglas
  41. Biochem. J. v.195 Isolation and properties lectins from mistletoe Franz, H.;P. Ziska;A. Kindt
  42. Proc. Natl. Acad. Sci. U.S.A. v.57 Assays for Agglutination with Lectins Burger, M. M.
  43. Lectin in higher plant. Biochemistry of plant Lis, H.;N. Sharon
  44. Sci. Amer. v.1 Carbohydrates in cell recognition Walkins, A. H.
  45. Methods Enzymol. v.28 Gorse phytohemagglutinins Osawa, T.;I. Matsumoto
  46. Agr. Biol. Chem. v.49 Purification and some properties of the basic lectin from windged bean seeds Higuchi, M.;K. Iwai