Journal of Protein Chemistry

, Volume 20, Issue 1, pp 49–58 | Cite as

Kinetic and Structural Properties of Two Isoforms of Trypsin Isolated from the Viscera of Japanese Anchovy, Engraulis japonicus

  • Md. Nazmul Ahsan
  • Shugo Watabe
Article

Abstract

Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH4)2SO4 fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (kcat/Km) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25°C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of Km values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.

Anchovy trypsin isoform characterization S1 pocket 

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REFERENCES

  1. Ahsan, M. N., Funabara, D., and Watabe, S. (2000). Marine Biotechnol. (in press).Google Scholar
  2. Ásgeirsson, B., Fox, J. W., and Bjarnason, J. B. (1989). Eur.J.Biochem. 180, 85–94.Google Scholar
  3. Bergmeyer, H. U., Gawehn, K., and Grassi, M. (1974). In Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Academic Press, New York, pp. 1013–1024.Google Scholar
  4. Bradford, M. M. (1976). Anal.Biochem. 72, 248–254.Google Scholar
  5. Britton, H. T. S. and Robinson, R. A. (1931). J.Chem.Soc. 1931, 1456–1462.Google Scholar
  6. Cohen, T., Gertler, A., and Birk, Y. (1981). Comp.Biochem.Physiol. 69B, 639–646.Google Scholar
  7. Davis, B. J. (1964). Ann.N.Y.Acad.Sci. 121, 404–427.Google Scholar
  8. Erlanger, B. F., Kokowsky, N., and Cohen, W. (1961). Arch.Biochem.Biophys. 95, 271–278.Google Scholar
  9. Fletcher, T. S., Alhadeff, M., Craik, C. S., and Largman, C. (1987). Biochemistry 26, 3081–3086.Google Scholar
  10. Gabel, D. and Kasche, V. (1973). Acta Chem.Scand. 27, 1971–1981.Google Scholar
  11. Genicot, S., Feller, G., and Gerday, Ch. (1988). Comp.Biochem.Physiol. 90B, 601–609.Google Scholar
  12. Genicot, S., Rentier-Delrue, F., Edwards, D., Vanbeeumen, J., and Gerday, C. (1996). Biochim.Biophys.Acta 1298, 45–57.Google Scholar
  13. Gildberg, A. and Overbo, K. (1990). Comp.Biochem.Physiol. 97B, 775–782.Google Scholar
  14. Gorfe, A. A., Brandsdal, B. O., Leiros, H. S., Helland, R., and Smalås A. O. (2000). Proteins 40, 207–217.Google Scholar
  15. Heu, M. S., Kim, H. R., and Pyeun, J. H. (1995). Comp.Biochem.Physiol. 112B, 557–567.Google Scholar
  16. Heussen, C. and Dowdle, E. B. (1980). Anal.Biochem. 102, 196–202.Google Scholar
  17. Hjelmeland, K. and Raa, J. (1982). Comp.Biochem.Physiol. 71B, 557–562.Google Scholar
  18. Hochachka, P. W. and Somero, G. N. (1984). In Biochemical Adaptation (Hochachka, P. W., and Somero, G. N., eds.), Princeton University Press, Princeton, New Jersey, pp. 377–388.Google Scholar
  19. Kim, H. R., Meyers, S. P., and Godber J. S. (1992). Comp.Biochem.Physiol. 103B, 391–398.Google Scholar
  20. Kim, H. R., Meyers, S. P., Pyeun, J. H., and Godber, J. S. (1994). Comp.Biochem.Physiol. 127B, 337–346.Google Scholar
  21. Laemmli, U. K. (1970). Nature 227, 680–685.Google Scholar
  22. Lam, W., Coast, G. M., and Rayne, R. C. (2000). Insect Biochem.Mol.Biol. 30, 85–94.Google Scholar
  23. Low, P. S. and Somero, G. N. (1974). Comp.Biochem.Physiol. 49B, 307–312.Google Scholar
  24. Mares-Guia, M. and Shaw, E. (1965). J.Biol.Chem. 240, 1579–1585.Google Scholar
  25. Martinez, A. and Serra, J. L. (1989). Comp.Biochem.Physiol. 93B, 61–66.Google Scholar
  26. Martinez, A., Olsen R. L., and Serra, J. (1988). Comp.Biochem.Physiol. 91B, 677–684.Google Scholar
  27. Matsudaira, P. (1987). J.Biol.Chem. 262, 10035–10038.Google Scholar
  28. Mihalyi, E. (1978). In Application of Proteolytic Enzymes to Protein Structure Studies (2nd ed.), CRC Press, Boca Raton, Florida, pp. 57–59.Google Scholar
  29. Murakami, K. and Noda, M. (1981). Biochim.Biophys.Acta 658, 17–26.Google Scholar
  30. Outzen, H., Berglund, G. I., Smalås, A. O., and Willassen, N. P. (1996). Comp.Biochem.Physiol. 115B, 33–45.Google Scholar
  31. Overnell, J. (1973). Comp.Biochem.Physiol. 46B, 519–531.Google Scholar
  32. Pollock, M. R. (1965). Biochem.J. 94, 666–675.Google Scholar
  33. Pyeun, J. H., Kim, H. R., and Godber J. S. (1990). Bull.Korean Fish.Soc. 23, 12–20.Google Scholar
  34. Raae, A. J. and Walther, W. T. (1989). Comp.Biochem.Physiol. 93B, 317–324.Google Scholar
  35. Rick, W. (1974). In Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Academic Press, New York, pp. 1013–1024.Google Scholar
  36. Robinson, C. N., Tye, W. R., Neurath, H., and Walsh, A. K. (1971). Biochemistry 10, 2743–2747.Google Scholar
  37. Rypniewski, W., Perrakis, A., Vorgias, C. E., and Wilson, K. S. (1994). Protein Eng. 7, 57–64.Google Scholar
  38. Schroeder, D. D. and Shaw, E. (1968). J.Biol.Chem. 243, 2943–2949.Google Scholar
  39. Sekizaki, H., Itoh, K., Murakami, M., Toyota, E., and Tanizawa K. (2000). Comp.Biochem.Physiol. 127B, 337–346.Google Scholar
  40. Simpson, B. K. and Haard, N. F. (1984a). Can.J.Biochem.Cell Biol. 62, 894–900.Google Scholar
  41. Simpson, B. K. and Haard, N. F. (1984b). Comp.Biochem.Physiol. 79B, 613–622.Google Scholar
  42. Simpson, B. K. and Haard, N. F. (1987). In Food Biotechnology (Knorr, D., ed.), Marcel Dekker, New York, pp. 495–527.Google Scholar
  43. Smalås, A. O., Heimstad, E. S., Hordvik, A., Willassen, N. P., and Male, R. (1994). Proteins 20, 149–166.Google Scholar
  44. Smith, R. L. and Shaw, E. (1969). J.Biol.Chem. 244, 4704–4712.Google Scholar
  45. Somero, G. N. (1978). Rev.Ecol.Syst. 9, 1–29.Google Scholar
  46. Szabo, E., Bocskei, Z., Naray-Szabo, G., and Graf, L. (1999). Eur.J.Biochem. 263, 20–26.Google Scholar
  47. Timasheff, S. N. (1969). Arch.Biochem.Biophys. 132, 165–169.Google Scholar
  48. Walker, J. M. (1994). In Basic Protein and Peptide Protocols (Walker J. M., ed.), Humana Press, Totowa, New Jersey, pp. 9–16.Google Scholar
  49. Winter, W. P. and Neurath, H. (1970). Biochemistry 9, 4673–4679.Google Scholar
  50. Zendzian, E. N. and Barnard, E. A. (1976). Arch.Biochem.Biophys. 122, 699–713.Google Scholar

Copyright information

© Plenum Publishing Corporation 2001

Authors and Affiliations

  • Md. Nazmul Ahsan
    • 1
  • Shugo Watabe
    • 1
  1. 1.Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life SciencesUniversity of TokyoTokyoJapan

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