Biochem J. 1985 Oct 15;231(2):451-7. doi: 10.1042/bj2310451.

Binding of proteinases to human alpha 2-macroglobulin with its thioester bonds cleaved by methylamine in the presence of a thiol-group-cyanylating reagent.

The Biochemical journal

I Björk

PMID: 2415116 PMCID: PMC1152767 DOI: 10.1042/bj2310451
Free PMC Article

Abstract

After cleavage of the thioester bonds of human alpha 2-macroglobulin (alpha 2M) by methylamine, the inhibitor undergoes an extensive conformational change and loses its ability to bind proteinases. In contrast, similar cleavage in the presence of dinitrophenyl thiocyanate, a reagent that cyanylates the liberated thiol groups, does not change the mobility of alpha 2M in gel electrophoresis, and the inhibitor also retains activity [Van Leuven, Marynen, Cassiman & Van den Berghe (1982) Biochem. J. 203, 405-411]. Analyses in this work show that also the spectroscopic properties of alpha 2M are essentially unperturbed under these conditions. These observations are consistent with the major change of the conformation of the protein having been arrested by the cyanylation reaction. However, several functional properties of the protein are altered, indicating that a limited conformational change does occur. The apparent stoichiometry of binding of trypsin is thus decreased to about 0.5 mol of enzyme/mol of alpha 2M. Nevertheless trypsin induces a similar conformational change in all molecules of the modified inhibitor as that induced in untreated alpha 2M. This behaviour indicates a similar mode of binding of the enzyme to the modified alpha 2M as to intact alpha 2M, but also a high extent of non-productive activation of binding sites in the modified inhibitor. A further difference to untreated alpha 2M is that most of the bound trypsin molecules react considerably faster with soya-bean trypsin inhibitor. The rate of inhibition of thrombin is also greatly decreased, and the modified inhibitor is more sensitive than untreated alpha 2M to proteolysis at sites outside the 'bait' region. The properties of the cyanylated human alpha 2M are thus similar to those of bovine alpha 2M in which the thioester bonds have been cleaved by methylamine in the absence of the cyanylating reagent [Björk, Lindblom & Lindahl (1985) Biochemistry 24, 2653-2660]. These results indicate that the thioester bonds of human and bovine alpha 2M are not required as such for the stability of the gross conformation of the protein or for the binding of proteinases. Nevertheless they participate directly in maintaining certain structural features, similar in the two inhibitors, that are necessary for full proteinase-binding ability. Disruption of these structures leads to a slower and less efficient trapping of the enzymes.

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References

1. Biochemistry. 1984 Jul 3;23(14):3115-24 - PubMed
2. Biochemistry. 1984 Jun 5;23(12):2802-7 - PubMed
3. Acta Chem Scand. 1966;20(8):2299-300 - PubMed
4. Biochim Biophys Acta. 1968 Jan 22;154(1):228-31 - PubMed
5. J Biol Chem. 1969 Aug 25;244(16):4406-12 - PubMed
6. J Biol Chem. 1969 Sep 25;244(18):4989-94 - PubMed
7. Biochemistry. 1971 Jul 6;10(14):2743-7 - PubMed
8. Biochem J. 1972 Mar;127(1):187-97 - PubMed
9. J Exp Med. 1973 Sep 1;138(3):508-21 - PubMed
10. Biochem J. 1973 Aug;133(4):709-24 - PubMed
11. Biochem Biophys Res Commun. 1975 Aug 4;65(3):927-30 - PubMed
12. Biochem Biophys Res Commun. 1975 Sep 16;66(2):482-9 - PubMed
13. Clin Chim Acta. 1976 Jan 2;66(1):1-7 - PubMed
14. Methods Enzymol. 1976;45:639-52 - PubMed
15. Biochem J. 1978 Jul 1;173(1):27-38 - PubMed
16. J Biol Chem. 1979 Jan 10;254(1):88-94 - PubMed
17. J Biol Chem. 1979 Jun 10;254(11):4452-6 - PubMed
18. Biochem J. 1979 Aug 1;181(2):401-18 - PubMed
19. Anal Biochem. 1979 Nov 1;99(2):415-20 - PubMed
20. J Biol Chem. 1981 Jan 25;256(2):547-50 - PubMed
21. FEBS Lett. 1980 Dec 1;121(2):275-9 - PubMed
22. FEBS Lett. 1981 Jan 12;123(1):145-8 - PubMed
23. FEBS Lett. 1981 May 18;127(2):167-73 - PubMed
24. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2235-9 - PubMed
25. J Biol Chem. 1981 Aug 10;256(15):7954-7 - PubMed
26. FEBS Lett. 1981 Jun 1;128(1):123-6 - PubMed
27. J Biol Chem. 1981 Oct 25;256(20):10409-14 - PubMed
28. J Biol Chem. 1981 Nov 10;256(21):10934-40 - PubMed
29. Biochem J. 1981 May 1;195(2):453-61 - PubMed
30. Eur J Biochem. 1982 Mar 1;122(3):663-6 - PubMed
31. Biochem J. 1982 May 1;203(2):405-11 - PubMed
32. Biochim Biophys Acta. 1982 Aug 10;705(3):306-14 - PubMed
33. Biochem Biophys Res Commun. 1982 Aug 31;107(4):1243-51 - PubMed
34. Biochemistry. 1982 Sep 14;21(19):4550-6 - PubMed
35. Biochem J. 1982 Nov 1;207(2):347-56 - PubMed
36. Arch Biochem Biophys. 1983 Feb 15;221(1):261-70 - PubMed
37. Biochemistry. 1983 Feb 1;22(3):536-46 - PubMed
38. Biochem J. 1983 Jan 1;209(1):99-105 - PubMed
39. Annu Rev Biochem. 1983;52:655-709 - PubMed
40. Biochemistry. 1983 Jul 19;22(15):3647-53 - PubMed
41. J Biol Chem. 1983 Dec 10;258(23):14682-5 - PubMed
42. Biochem J. 1984 Jan 1;217(1):303-8 - PubMed
43. Biochem Biophys Res Commun. 1984 Jan 30;118(2):691-5 - PubMed
44. J Biol Chem. 1984 Apr 10;259(7):4458-62 - PubMed
45. Biochem J. 1980 Jun 1;187(3):695-701 - PubMed
46. Biochemistry. 1985 May 21;24(11):2653-60 - PubMed

Substances

• Dinitrobenzenes
• Methylamines
• Nitrobenzenes
• Peptide Fragments
• Trypsin Inhibitors
• alpha-Macroglobulins
• methylamine
• Trypsin
• Thrombin
• 2,4-dinitrothiocyanatobenzene

MeSH terms

• Dinitrobenzenes / metabolism
• Electrophoresis, Polyacrylamide Gel
• Kinetics
• Methylamines / metabolism
• Nitrobenzenes / metabolism
• Peptide Fragments / metabolism
• Protein Binding
• Protein Conformation
• Spectrophotometry
• Thrombin / antagonists & inhibitors
• Trypsin / metabolism
• Trypsin Inhibitors / pharmacology
• alpha-Macroglobulins / metabolism

Publication Types

• Journal Article
• Research Support, Non-U.S. Gov't

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