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Enteropeptidase

Structure, function, and application in biotechnology

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Abstract

A preparative method for purification of enteropeptidase (enterokinase) (EC 3. 4. 21.9) is developed. A highly purified form of this enzyme is stabilized by calcium ions and does not contain any other proteolytic enzyme contaminations. These enteropeptidase preparations were successfully used for cleavage of a variety of fusion proteins containing the tetraaspartyl-lysyl sequence in an arbitrary position on the polypeptide chain. A series of substrates was methodically studied, which resulted in the suggestion that the peptide and fusion protein substrates (K m -200μM and 125μM, respectively) were bound to the enzyme through the linker (Asp)4 Lys at the binding site on the light chain of enteropeptidase. Much more efficient hydrolysis of the natural substrate trypsinogen (K m=2.4μM) testifies to a significant contribution of other sites of the substrate and the enzyme in productive binding Avariation in the enzyme's uniquespecificity wasshown to be a result of the autolysis caused by the loss of calcium ions; the cleavage sites were determined. The truncated enzyme containing the C-terminal fragment 466–800 of its heavy chain and the intact light chain does not distinguish the natural substrate trypsinogen, fusion protein, or peptide substrates. These results suggest that the N-terminal fragment 118–465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.

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Abbreviations

BPI:

basic bovine protease inhibitor

STI:

soybean trypsin inhibitor

MPG-PA-2000:

porous glass chemically coated with poly (4-nitrophenyl acrylate)

OG:

n-octyl-β-d-glucopyranoside

BAPNA:

α-N-bensotyl-dl-arginine 4-nitroanilide

GD4K-NA:

Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide

Gdn-Bz-ONp:

4′-guanidinobenzoate 4-nitrophenyl ester

Gdn-Bz-OMum:

4′-guanidinobenzoate 4-methylumbelliferil ester

References

  1. Kunitz, M. (1939), J. Gen. Physiol. 2, 429–446.

    Article  Google Scholar 

  2. Liepnieks, J. J. and Light, A. (1979), J. Biol. Chem. 254, 1677–1683

    CAS  Google Scholar 

  3. Light, A., and Fonseca, P. (1984), J. Biol. Chem. 259, 13195–13198.

    CAS  Google Scholar 

  4. Light, A., Savithri, H. S., and Liepnieks, J. J. (1980), Anal. Biochem. 106, 199–206.

    Article  CAS  Google Scholar 

  5. Kitamoto, Y., Yuan, X., Wu, Q., McCourt, D. W., and Sadler, J. E. (1994), Proc. Natl. Acad. Sci. USA 91, 7588–7592.

    Article  CAS  Google Scholar 

  6. Matsushima, M., Ichinose, M., Yahagi, N., Kakei, N., Tsukada, S., Miki, K., et al. (1994), J. Biol. Chem. 269, 19,976–19,982.

    CAS  Google Scholar 

  7. Kitamoto, Y., Veile, R. A., Donis-Keller, H., and Sadler, J. E. (1995), Biochemistry 34, 562–568.

    Article  Google Scholar 

  8. Yahagi, N., Ichinose, M., Matsushima, M., Matsubara, Y., Miki, K., Kurokawa, K., et al. (1996), Biochem. Biophys. Res. Commun. 219, 806–812.

    Article  CAS  Google Scholar 

  9. LaVallie, E. R., Rehemtulla, A., Racie, L. A., DiBlasio, E. A., Ferenz, C., Grant, K. L., Light, A., and McCoy, J. M. (1993), J. Biol. Chem. 268, 23,311–23,316.

    CAS  Google Scholar 

  10. Collins-Racie, L. A., McColgan, J. M., Grant, K. L., DiBlasio-Smith, E. A., McCoy, J. M., and La Vallie, E. R. (1995), Bio/Technology 13, 982–987.

    Article  CAS  Google Scholar 

  11. Vozza, L. A., Wittwer, L., Higgins, D. R., Purcell, T. J., Bergseid, M., Collins-Racie, L. A., LaVallie, E. R., and Hoeffler, J. P. (1996), Bio/Technology 14, 77–81.

    Article  CAS  Google Scholar 

  12. Lu, D., Yuan, X., Zheng, X., and Sadler, J. E. (1997), J. Biol. Chem. 272, 31,293–31,300.

    CAS  Google Scholar 

  13. Guy, O., Bartelt, D. C., Amic, J., Colomb, E., and Figarella, C. (1976), FEBS Lett. 62, 150–153.

    Article  CAS  Google Scholar 

  14. LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant, K. L., Schendel, P. F., and McCoy, J. M. (1993), Bio/Technology 11, 187–193.

    Article  CAS  Google Scholar 

  15. Dykes, C. W., Bookless, A. B., Coomber, B. A., Noble, S. A., Humber, D. C., and Hobden, A. N. (1988), Eur. J. Biochem. 174, 411–416.

    Article  CAS  Google Scholar 

  16. Hopp, T. P., Prickett, K. S., Libby, R. T., March, C. J., Cerretti, D. P., Urdal, D. L., and Conlon, P. G. (1988), Bio/Technology 6, 1204–1210.

    Article  CAS  Google Scholar 

  17. Uegaki, K., Nemoto, N., Shimizu, M., Wada, T., Kyogoku, Y., and Kobayashi, Y. (1996), FEBS Lett. 379, 47–50.

    Article  CAS  Google Scholar 

  18. March, S. C., Parikh, I., and Cuatrecasas, P. (1974), Anal. Biochem. 60, 149–152.

    Article  CAS  Google Scholar 

  19. Kudryavtseva, N. E., Zhigis, L. S., Zubov, V. G., Vulfson, A. N., Maltsev, K. V., and Rumsh, L. D. (1995), Khim. Pharm. Zh. 1, 61–63 (in Russian).

    Google Scholar 

  20. Mikhailova, A. G., Evtyukova, N. G., Chupova, L. A., and Rumsh, L. D. (1998), Vopr. Med. Khim., 4, in press (in Russian).

  21. Zamolodchikova, T. S., Vorotyntseva, T. I., and Antonov, V. K. (1995), Eur. J. Biochem. 227, 866–872.

    Article  CAS  Google Scholar 

  22. Bradford, M. (1976), Anal. Biochem. 72, 248–254.

    Article  CAS  Google Scholar 

  23. Whitaker, J. R. and Granum, P. E. (1980), Anal. Biochem. 109, 156–159.

    Article  CAS  Google Scholar 

  24. Kassel, B. (1970), Methods Enzymol. 19, 844–853.

    Article  Google Scholar 

  25. Chase, T., Jr., and Shaw, E. (1970), Methods Enzymol. 19, 20–27.

    CAS  Google Scholar 

  26. Laemmli, U. K. (1970), Nature 227, 680–685.

    Article  CAS  Google Scholar 

  27. Mikkailova, A. G., Shibanova, E. D., Rumsh, L. D., and Antonov, V. K. (1994), Bioorg. Khim. 20, 883–893 (in Russian).

    Google Scholar 

  28. Grant, D. A. W. and Hermon-Taylor, J. (1979), Biochim. Biophys. Acta 567, 207–215.

    CAS  Google Scholar 

  29. Eisenthal, R. and Cornish-Bowden, A. (1974), Biochem. J. 139, 715–720.

    CAS  Google Scholar 

  30. Mikhailova, A. G. and Rumsh, L. D. (1998), Bioorg. Khim. 24, 282–287 (in Russian).

    CAS  Google Scholar 

  31. Zamolodchikova, T. S., Sokolova, E. A., Alexandrov, S. L., Mikkaleva, I. I., Prudchenko, I. A., Morozov, I. A., et al. (1997), Eur. J. Biochem. 249, 612–621.

    Article  CAS  Google Scholar 

  32. Dobrynin, V. N., Boldyreva, E. F., Filippov, S. A., Chuvpilo, S. A., Korobko, V. G., Vorotyntseva, T. I., et al. (1987) Bioorg. Khim. 13, 119–121 (in Russian).

    CAS  Google Scholar 

  33. Kutusov, M. A., Schmuckler, B. E., Suslov, O. N., Zargarov, A. A., and Abdulaev, N. G. (1992), Bioorg. Khim. 18, 119–121 (in Russian).

    Google Scholar 

  34. Gaidarov, I. O., Suslov, O. N., Ovchinnikova, T. V., and Abdulaev, N. G. (1994), Bioorg. Khim. 20, 367–381 (in Russian).

    CAS  Google Scholar 

  35. Dergousova, N. I., Amerik, A. Yu., Volynskaya, A. M., and Rumsh, L. D. (1996), Appl. Biochem. Biotechnol. 61, 97–106.

    CAS  Google Scholar 

  36. Kostanyan, I. A., Astapova, M. V., Starovoytova, S. M., Dranitsina, S. M., and Lipkin, V. M. (1994), FEBS Lett. 356, 327–329.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP-7, 117871, Moscow, Russia

    Anna G. Mikhailova & Lev D. Rumsh

Authors
  1. Anna G. Mikhailova
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  2. Lev D. Rumsh
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Correspondence to Anna G. Mikhailova.

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Mikhailova, A.G., Rumsh, L.D. Enteropeptidase. Appl Biochem Biotechnol 88, 159–174 (2000). https://doi.org/10.1385/ABAB:88:1-3:159

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  • DOI: https://doi.org/10.1385/ABAB:88:1-3:159

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