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. 1985 Oct 15;231(2):417–423. doi: 10.1042/bj2310417

Kinetics of the reaction of thrombin and alpha 2-macroglobulin.

R D Feinman, A I Yuan, S R Windwer, D Wang
PMCID: PMC1152762  PMID: 2415115

Abstract

The kinetics of the reaction of alpha 2-macroglobulin (alpha 2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native alpha 2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester centre, and this may be suggestive evidence for a reactive alpha 2M centre that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several alpha 2M chains by the bound proteinase, providing further evidence that the very-high-Mr species seen on gels may arise from dimers of the alpha 2M molecule held together by covalent bonds to the enzyme.

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Selected References

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  1. Barrett A. J. Alpha 2-macroglobulin. Methods Enzymol. 1981;80(Pt 100):737–754. doi: 10.1016/s0076-6879(81)80056-0. [DOI] [PubMed] [Google Scholar]
  2. Barrett A. J., Brown M. A., Sayers C. A. The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule. Biochem J. 1979 Aug 1;181(2):401–418. doi: 10.1042/bj1810401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrett A. J., Starkey P. M. The interaction of alpha 2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem J. 1973 Aug;133(4):709–724. doi: 10.1042/bj1330709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Christensen U., Sottrup-Jensen L. Mechanism of alpha 2-macroglobulin-proteinase interactions. Studies with trypsin and plasmin. Biochemistry. 1984 Dec 18;23(26):6619–6626. doi: 10.1021/bi00321a052. [DOI] [PubMed] [Google Scholar]
  5. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  6. Erickson B. W., Khan S. A. Synthetic lactam and thiolactone models of protein metastable binding sites. Ann N Y Acad Sci. 1983;421:167–177. doi: 10.1111/j.1749-6632.1983.tb18107.x. [DOI] [PubMed] [Google Scholar]
  7. Feinman R. D., Wang D., Windwer S. R., Wu K. The role of enzyme lysyl amino groups in the reaction with alpha 2-macroglobulin. Ann N Y Acad Sci. 1983;421:178–187. doi: 10.1111/j.1749-6632.1983.tb18108.x. [DOI] [PubMed] [Google Scholar]
  8. Gonias S. L., Pizzo S. V. Characterization of functional human alpha 2-macroglobulin half-molecules isolated by limited reduction with dithiothreitol. Biochemistry. 1983 Feb 1;22(3):536–546. doi: 10.1021/bi00272a003. [DOI] [PubMed] [Google Scholar]
  9. Gonias S. L., Pizzo S. V. Chemical and structural modifications of alpha 2-macroglobulin: effects on receptor binding and endocytosis studied in an in vivo model. Ann N Y Acad Sci. 1983;421:457–471. doi: 10.1111/j.1749-6632.1983.tb18139.x. [DOI] [PubMed] [Google Scholar]
  10. Grassetti D. R., Murray J. F., Jr Determination of sulfhydryl groups with 2,2'- or 4,4'-dithiodipyridine. Arch Biochem Biophys. 1967 Mar;119(1):41–49. doi: 10.1016/0003-9861(67)90426-2. [DOI] [PubMed] [Google Scholar]
  11. Howard J. B. Reactive centers in alpha 2-macroglobulin. Ann N Y Acad Sci. 1983;421:69–80. doi: 10.1111/j.1749-6632.1983.tb18093.x. [DOI] [PubMed] [Google Scholar]
  12. Howard J. B., Vermeulen M., Swenson R. P. The temperature-sensitive bond in human alpha 2-macroglobulin is the alkylamine-reactive site. J Biol Chem. 1980 May 10;255(9):3820–3823. [PubMed] [Google Scholar]
  13. Khan S. A., Erickson B. W. An equilibrium model of the metastable binding sites of alpha 2-macroglobulin and complement proteins C3 and C4. J Biol Chem. 1982 Oct 25;257(20):11864–11867. [PubMed] [Google Scholar]
  14. Krebs G., Guinand S., Bréda C. Modèle d'interaction entre la trypsine et la alpha2-macroglobuline du sérum de Porc. C R Acad Sci Hebd Seances Acad Sci D. 1978 Apr 24;286(16):1219–1222. [PubMed] [Google Scholar]
  15. Kurecki T., Kress L. F., Laskowski M., Sr Purification of human plasma alpha 2 macroglobulin and alpha 1 proteinase inhibitor using zinc chelate chromatography. Anal Biochem. 1979 Nov 1;99(2):415–420. doi: 10.1016/s0003-2697(79)80026-3. [DOI] [PubMed] [Google Scholar]
  16. Salvesen G. S., Barrett A. J. Covalent binding of proteinases in their reaction with alpha 2-macroglobulin. Biochem J. 1980 Jun 1;187(3):695–701. doi: 10.1042/bj1870695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Salvesen G. S., Sayers C. A., Barrett A. J. Further characterization of the covalent linking reaction of alpha 2-macroglobulin. Biochem J. 1981 May 1;195(2):453–461. doi: 10.1042/bj1950453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sottrup-Jensen L., Hansen H. F., Christensen U. Generation and reactivity of "nascent" alpha 2-macroglobulin: localization of cross-links in alpha 2-macroglobulin-trypsin complex. Ann N Y Acad Sci. 1983;421:188–208. doi: 10.1111/j.1749-6632.1983.tb18109.x. [DOI] [PubMed] [Google Scholar]
  19. Sottrup-Jensen L., Petersen T. E., Magnusson S. A thiol-ester in alpha 2-macroglobulin cleaved during proteinase complex formation. FEBS Lett. 1980 Dec 1;121(2):275–279. doi: 10.1016/0014-5793(80)80361-9. [DOI] [PubMed] [Google Scholar]
  20. Straight D. L., McKee P. A. Characterization of thrombin binding to alpha 2-macroglobulin. J Biol Chem. 1984 Jan 25;259(2):1272–1278. [PubMed] [Google Scholar]
  21. Straight D. L., McKee P. A. Effect of protease binding by alpha 2-macroglobulin on intrinsic fluorescence. Biochemistry. 1982 Sep 14;21(19):4550–4556. doi: 10.1021/bi00262a006. [DOI] [PubMed] [Google Scholar]
  22. Van Leuven F., Cassiman J. J., Van den Berghe H. Functional modifications of alpha 2-macroglobulin by primary amines. Kinetics of inactivation of alpha 2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin. Biochem J. 1982 Jan 1;201(1):119–128. doi: 10.1042/bj2010119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Van Leuven F., Marynen P., Cassiman J. J., Van den Berghe H. Relation of internal thioesters to conformational change and receptor-recognition site in alpha 2-macroglobulin complexes. Biochem J. 1982 May 1;203(2):405–411. doi: 10.1042/bj2030405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Virca G. D., Travis J., Hall P. K., Roberts R. C. Purification of human alpha-2-macroglobulin by chromatography on Cibacron Blue Sepharose. Anal Biochem. 1978 Aug 15;89(1):274–278. doi: 10.1016/0003-2697(78)90750-9. [DOI] [PubMed] [Google Scholar]
  25. Wang D., Wu K., Feinman R. D. Alpha 2-macroglobulin-protease reactions: relationship of covalent bond formation, methylamine reactivity, and specific proteolysis. Arch Biochem Biophys. 1981 Oct 1;211(1):500–506. doi: 10.1016/0003-9861(81)90483-5. [DOI] [PubMed] [Google Scholar]
  26. Wang D., Yuan A. I., Feinman R. D. Covalent thrombin-alpha 2-macroglobulin complexes. Evidence for bivalent cross-linking of inhibitor chains by a single enzyme molecule. Biochemistry. 1984 Jun 5;23(12):2807–2811. doi: 10.1021/bi00307a042. [DOI] [PubMed] [Google Scholar]
  27. Wang D., Yuan A. I., Feinman R. D. Structure of alpha 2-macroglobulin-protease complexes. Ann N Y Acad Sci. 1983;421:90–97. doi: 10.1111/j.1749-6632.1983.tb18095.x. [DOI] [PubMed] [Google Scholar]
  28. Wong R. F., Windwer S. R., Feinman R. D. Interaction of thrombin and antithrombin. Reaction observed by intrinsic fluorescence measurements. Biochemistry. 1983 Aug 16;22(17):3994–3999. doi: 10.1021/bi00286a001. [DOI] [PubMed] [Google Scholar]
  29. Wu K., Wang D., Feinman R. D. Inhibition of proteases by alpha 2-macroglobulin. The role of lysyl amino groups of trypsin in covalent complex formation. J Biol Chem. 1981 Oct 25;256(20):10409–10414. [PubMed] [Google Scholar]
  30. van der Graaf F., Rietveld A., Keus F. J., Bouma B. N. Interaction of human plasma kallikrein and its light chain with alpha 2-macroglobulin. Biochemistry. 1984 Apr 10;23(8):1760–1766. doi: 10.1021/bi00303a027. [DOI] [PubMed] [Google Scholar]

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