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. 1992 Aug 15;286(Pt 1):165–171. doi: 10.1042/bj2860165

Characterization by rapid-kinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actinidin.

P Lindahl 1, M Nycander 1, K Ylinenjärvi 1, E Pol 1, I Björk 1
PMCID: PMC1133034  PMID: 1520264

Abstract

The interaction between five N-terminally truncated forms of chicken cystatin (starting at Leu-7, Leu-8, Gly-9, Ala-10 and Asp-15) and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The u.v. absorption, near-u.v. c.d. and fluorescence emission difference spectra for the interactions with papain were all similar to the corresponding spectra for intact cystatin. The second-order association rate constants at 25 degrees C, pH 7.4, I 0.15, for the binding of the truncated forms to papain varied about 2-fold, from 6 x 10(6) to 1.5 x 10(7) M-1.s-1, and were comparable to the value of 9.9 x 10(6) M-1.s-1 for intact cystatin. In contrast, the rate constants for the dissociation of the complexes with papain increased markedly with increasing extent of truncation, from 7.5 x 10(-6)s-1 for Leu7 cystatin (a truncated form of cystatin having Leu-7 as its N-terminal amino acid) to 1.6s-1 for Ala10-cystatin, whereas the dissociation rate constants for the latter form and Asp15-cystatin were similar. Consequently, the binding affinities between the truncated cystatins and papain decreased in an analogous manner, as was also shown for the interaction with actinidin by equilibrium measurements. Studies of the binding of the truncated cystatins to inactivated papains indicated that small substituents on the active-site cysteine of the enzyme can be accommodated in the complex without any loss of affinity when the N-terminal segment of the inhibitor is removed. Taken together, the results suggest that in the N-terminal region of chicken cystatin only residues preceding Ala-10 participate in the interaction with proteinases. Of these residues, Leu-7 and Leu-8 together account for about two-thirds of the unitary free energy of binding contributed by the N-terminal region, the relative importance of the two residues being dependent on the target proteinase. Both Gly-9 and residues N-terminal of Leu-7 further stabilize the interaction but contribute substantially smaller binding energies than do the two leucine residues.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abrahamson M., Mason R. W., Hansson H., Buttle D. J., Grubb A., Ohlsson K. Human cystatin C. role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase. Biochem J. 1991 Feb 1;273(Pt 3):621–626. doi: 10.1042/bj2730621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abrahamson M., Ritonja A., Brown M. A., Grubb A., Machleidt W., Barrett A. J. Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. J Biol Chem. 1987 Jul 15;262(20):9688–9694. [PubMed] [Google Scholar]
  3. Anastasi A., Brown M. A., Kembhavi A. A., Nicklin M. J., Sayers C. A., Sunter D. C., Barrett A. J. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem J. 1983 Apr 1;211(1):129–138. doi: 10.1042/bj2110129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Axén R., Porath J., Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature. 1967 Jun 24;214(5095):1302–1304. doi: 10.1038/2141302a0. [DOI] [PubMed] [Google Scholar]
  5. Barrett A. J., Kembhavi A. A., Brown M. A., Kirschke H., Knight C. G., Tamai M., Hanada K. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982 Jan 1;201(1):189–198. doi: 10.1042/bj2010189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Björk I., Alriksson E., Ylinenjärvi K. Kinetics of binding of chicken cystatin to papain. Biochemistry. 1989 Feb 21;28(4):1568–1573. doi: 10.1021/bi00430a022. [DOI] [PubMed] [Google Scholar]
  7. Björk I., Ylinenjärvi K. Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin. Biochemistry. 1990 Feb 20;29(7):1770–1776. doi: 10.1021/bi00459a016. [DOI] [PubMed] [Google Scholar]
  8. Björk I., Ylinenjärvi K. Interaction of chicken cystatin with inactivated papains. Biochem J. 1989 May 15;260(1):61–68. doi: 10.1042/bj2600061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bode W., Engh R., Musil D., Laber B., Stubbs M., Huber R., Turk V. Mechanism of interaction of cysteine proteinases and their protein inhibitors as compared to the serine proteinase-inhibitor interaction. Biol Chem Hoppe Seyler. 1990 May;371 (Suppl):111–118. [PubMed] [Google Scholar]
  10. Bode W., Engh R., Musil D., Thiele U., Huber R., Karshikov A., Brzin J., Kos J., Turk V. The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J. 1988 Aug;7(8):2593–2599. doi: 10.1002/j.1460-2075.1988.tb03109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brocklehurst K., Baines B. S., Malthouse J. P. Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes. Biochem J. 1981 Sep 1;197(3):739–746. doi: 10.1042/bj1970739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Brocklehurst K., Carlsson J., Kierstan M. P., Crook E. M. Covalent chromatography. Preparation of fully active papain from dried papaya latex. Biochem J. 1973 Jul;133(3):573–584. doi: 10.1042/bj1330573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Burke D. E., Lewis S. D., Shafer J. A. A two-step procedure for purification of papain from extract of papaya latex. Arch Biochem Biophys. 1974 Sep;164(1):30–36. doi: 10.1016/0003-9861(74)90004-6. [DOI] [PubMed] [Google Scholar]
  14. Buttle D. J., Kembhavi A. A., Sharp S. L., Shute R. E., Rich D. H., Barrett A. J. Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex. Biochem J. 1989 Jul 15;261(2):469–476. doi: 10.1042/bj2610469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Buttle D. J., Ritonja A., Dando P. M., Abrahamson M., Shaw E. N., Wikstrom P., Turk V., Barrett A. J. Interactions of papaya proteinase IV with inhibitors. FEBS Lett. 1990 Mar 12;262(1):58–60. doi: 10.1016/0014-5793(90)80153-a. [DOI] [PubMed] [Google Scholar]
  16. Carne A., Moore C. H. The amino acid sequence of the tryptic peptides from actinidin, a proteolytic enzyme from the fruit of Actinidia chinensis. Biochem J. 1978 Jul 1;173(1):73–83. doi: 10.1042/bj1730073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fossum K., Whitaker J. R. Ficin and papain inhibitor from chicken egg white. Arch Biochem Biophys. 1968 Apr;125(1):367–375. doi: 10.1016/0003-9861(68)90672-3. [DOI] [PubMed] [Google Scholar]
  18. Husain S. S., Lowe G. Completion of the amino acid sequence of papain. Biochem J. 1969 Sep;114(2):279–288. doi: 10.1042/bj1140279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lindahl P., Alriksson E., Jörnvall H., Björk I. Interaction of the cysteine proteinase inhibitor chicken cystatin with papain. Biochemistry. 1988 Jul 12;27(14):5074–5082. doi: 10.1021/bi00414a019. [DOI] [PubMed] [Google Scholar]
  21. Machleidt W., Thiele U., Laber B., Assfalg-Machleidt I., Esterl A., Wiegand G., Kos J., Turk V., Bode W. Mechanism of inhibition of papain by chicken egg white cystatin. Inhibition constants of N-terminally truncated forms and cyanogen bromide fragments of the inhibitor. FEBS Lett. 1989 Jan 30;243(2):234–238. doi: 10.1016/0014-5793(89)80135-8. [DOI] [PubMed] [Google Scholar]
  22. McDowall M. A. Anionic proteinase from Actinidia chinensis. Preparation and properties of the crystalline enzyme. Eur J Biochem. 1970 Jun;14(2):214–221. doi: 10.1111/j.1432-1033.1970.tb00280.x. [DOI] [PubMed] [Google Scholar]
  23. Nicklin M. J., Barrett A. J. Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin. Biochem J. 1984 Oct 1;223(1):245–253. doi: 10.1042/bj2230245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nordenman B., Björk I. Binding of low-affinity and high-affinity heparin to antithrombin. Ultraviolet difference spectroscopy and circular dichroism studies. Biochemistry. 1978 Aug 8;17(16):3339–3344. doi: 10.1021/bi00609a026. [DOI] [PubMed] [Google Scholar]
  25. Nycander M., Björk I. Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site. Biochem J. 1990 Oct 1;271(1):281–284. doi: 10.1042/bj2710281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ohkubo I., Kurachi K., Takasawa T., Shiokawa H., Sasaki M. Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen. Biochemistry. 1984 Nov 20;23(24):5691–5697. doi: 10.1021/bi00319a005. [DOI] [PubMed] [Google Scholar]
  27. Popović T., Brzin J., Ritonja A., Turk V. Different forms of human cystatin C. Biol Chem Hoppe Seyler. 1990 Jul;371(7):575–580. doi: 10.1515/bchm3.1990.371.2.575. [DOI] [PubMed] [Google Scholar]
  28. Rawlings N. D., Barrett A. J. Evolution of proteins of the cystatin superfamily. J Mol Evol. 1990 Jan;30(1):60–71. doi: 10.1007/BF02102453. [DOI] [PubMed] [Google Scholar]
  29. Roberts D. D., Lewis S. D., Ballou D. P., Olson S. T., Shafer J. A. Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry. 1986 Sep 23;25(19):5595–5601. doi: 10.1021/bi00367a038. [DOI] [PubMed] [Google Scholar]
  30. Schwabe C., Anastasi A., Crow H., McDonald J. K., Barrett A. J. Cystatin. Amino acid sequence and possible secondary structure. Biochem J. 1984 Feb 1;217(3):813–817. doi: 10.1042/bj2170813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stubbs M. T., Laber B., Bode W., Huber R., Jerala R., Lenarcic B., Turk V. The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J. 1990 Jun;9(6):1939–1947. doi: 10.1002/j.1460-2075.1990.tb08321.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thiele U., Assfalg-Machleidt I., Machleidt W., Auerswald E. A. N-terminal variants of recombinant stefin B: effect on affinity for papain and cathepsin B. Biol Chem Hoppe Seyler. 1990 May;371 (Suppl):125–136. [PubMed] [Google Scholar]

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