Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1983 Jan 1;209(1):99–105. doi: 10.1042/bj2090099

Ligand binding, conformational change and plasma elimination of human, mouse and rat alpha-macroglobulin proteinase inhibitors.

S L Gonias, A E Balber, W J Hubbard, S V Pizzo
PMCID: PMC1154060  PMID: 6189480

Abstract

Rat alpha 1-macroglobulin (alpha 1M), rat alpha 2-macroglobulin (alpha 2M) migrated as single bands on non-denaturing gels when purified by the methods described. All three proteins demonstrated increased mobility after reaction with trypsin. A single saturable pathway rapidly cleared complexes of trypsin and the alpha-macroglobulins of mouse, rat and human from the circulation of mice. None of the native alpha-macroglobulins competed for clearance with the trypsin complexes. [14C]Methylamine incorporation was 4.1, 3.9, 2.6 and 3.2 mol/mol of proteinase inhibitor for human alpha 2M, rat alpha 1M, rat alpha 2M and mouse alpha 2M, respectively. Only rat alpha 2M, the acute-phase alpha-macroglobulin studied, showed no evidence of conformational change when subjected to electrophoresis after reaction with methylamine. The clearance of rat alpha 2M-methylamine was comparable with that of the native molecule. The other alpha-macroglobulin-methylamine complexes cleared faster than the inhibitors that had not reacted. Rat alpha 2M and rat alpha 2M-methylamine bound equivalent quantities of 1251-labelled trypsin (1.01 and 0.96 mol/mol respectively). The soya-bean trypsin inhibitor-resistant esterolytic activity of trypsin bound to rat alpha 2M-methylamine was approx. 90% suppressed compared with proteinase bound to native rat alpha 2M. This suppression was not due to a change in the affinity of soya-bean trypsin inhibitor for the complex. Reaction of rat alpha 2M-methylamine with trypsin resulted in a 'slow' to 'fast' electrophoretic conversion of the proteinase inhibitor, and exposure of the signal on the alpha 2M that causes the complex to clear from the murine circulation.

Full text

PDF
99

Images in this article

101Fig. 1.
on p.101
103Fig. 6.
on p.103

Selected References

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

  1. 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]
  2. 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]
  3. Chase T., Jr, Shaw E. p-Nitrophenyl-p'-guanidinobenzoate HCl: a new active site titrant for trypsin. Biochem Biophys Res Commun. 1967 Nov 30;29(4):508–514. doi: 10.1016/0006-291x(67)90513-x. [DOI] [PubMed] [Google Scholar]
  4. DARCY D. A. Immunological discrimination between the blood of normal and of tumour-bearing rats. Nature. 1955 Oct 1;176(4483):643–644. doi: 10.1038/176643a0. [DOI] [PubMed] [Google Scholar]
  5. David G. S., Reisfeld R. A. Protein iodination with solid state lactoperoxidase. Biochemistry. 1974 Feb 26;13(5):1014–1021. doi: 10.1021/bi00702a028. [DOI] [PubMed] [Google Scholar]
  6. Debanne M. T., Bell R., Dolovich J. Uptake of proteinase-alpha-macroglobulin complexes by macrophages. Biochim Biophys Acta. 1975 Dec 5;411(2):295–304. doi: 10.1016/0304-4165(75)90309-8. [DOI] [PubMed] [Google Scholar]
  7. Ganrot P. O. Determination of alpha-2-macroglobulin as trypsin-protein esterase. Clin Chim Acta. 1966 Oct;14(4):493–501. doi: 10.1016/0009-8981(66)90037-4. [DOI] [PubMed] [Google Scholar]
  8. Gauthier F., Mouray H. Rat alpha2 acute-phase macroglobulin. Isolation and physicochemical properties. Biochem J. 1976 Dec 1;159(3):661–665. doi: 10.1042/bj1590661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gonias S. L., Reynolds J. A., Pizzo S. V. Physical properties of human alpha 2-macroglobulin following reaction with methylamine and trypsin. Biochim Biophys Acta. 1982 Aug 10;705(3):306–314. doi: 10.1016/0167-4838(82)90252-7. [DOI] [PubMed] [Google Scholar]
  10. Gordon A. H. The alpha macroglobulins of rat serum. Biochem J. 1976 Dec 1;159(3):643–650. doi: 10.1042/bj1590643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Greene N. D., Damian R. T., Hubbard W. J. The identification of alpha-2-macroglobulin in the mouse. Biochim Biophys Acta. 1971 Jun 29;236(3):659–663. doi: 10.1016/0005-2795(71)90252-2. [DOI] [PubMed] [Google Scholar]
  12. Hall P. K., Roberts R. C. Physical and chemical properties of human plasma alpha2-macroglobulin. Biochem J. 1978 Jul 1;173(1):27–38. doi: 10.1042/bj1730027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harpel P. C., Hayes M. B., Hugli T. E. Heat-induced fragmentation of human alpha 2-macroglobulin. J Biol Chem. 1979 Sep 10;254(17):8669–8678. [PubMed] [Google Scholar]
  14. Harpel P. C. Studies on human plasma alpha 2-macroglobulin-enzyme interactions. Evidence for proteolytic modification of the subunit chain structure. J Exp Med. 1973 Sep 1;138(3):508–521. doi: 10.1084/jem.138.3.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hudig D., Sell S. Isolation, characterization and radioimmunoassay of rat alpha macrofetoprotein (acute phase A2 macroglobulin). Mol Immunol. 1979 Aug;16(8):547–554. doi: 10.1016/0161-5890(79)90116-0. [DOI] [PubMed] [Google Scholar]
  16. Imber M. J., Pizzo S. V. Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin. J Biol Chem. 1981 Aug 10;256(15):8134–8139. [PubMed] [Google Scholar]
  17. Kaplan J., Ray F. A., Keogh E. A. Recognition of nucleophile-treated alpha 2-macroglobulin by the alveolar macrophage alpha-macroglobulin . protease complex receptor. J Biol Chem. 1981 Aug 10;256(15):7705–7707. [PubMed] [Google Scholar]
  18. 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]
  19. Nelles L. P., Hall P. K., Roberts R. C. Human alpha-2-macroglobulin. Studies on the electrophoretic heterogeneity. Biochim Biophys Acta. 1980 May 29;623(1):46–56. doi: 10.1016/0005-2795(80)90006-9. [DOI] [PubMed] [Google Scholar]
  20. Ohlsson K. Isolation and partial characterization of two related trypsin binding alpha-macroglobulins of dog plasma. Biochim Biophys Acta. 1971 Apr 27;236(1):84–91. doi: 10.1016/0005-2795(71)90153-x. [DOI] [PubMed] [Google Scholar]
  21. PICARD J. J., HEREMANS J. F. Studies on alpha2-macroblobulin. I. A method for the isolation of rabbit alpha2-macroglobulin. Biochim Biophys Acta. 1963 Jun 4;71:554–561. doi: 10.1016/0006-3002(63)91127-2. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Sottrup-Jensen L., Hansen H. F., Mortensen S. B., Petersen T. E., Magnusson S. Sequence location of the reactive thiol ester in human alpha 2-macroglobulin. FEBS Lett. 1981 Jan 12;123(1):145–148. doi: 10.1016/0014-5793(81)80039-7. [DOI] [PubMed] [Google Scholar]
  24. Sottrup-Jensen L., Lønblad P. B., Stepanik T. M., Petersen T. E., Magnusson S., Jörnvall H. Primary structure of the 'bait' region for proteinases in alpha 2-macroglobulin. Nature of the complex. FEBS Lett. 1981 May 18;127(2):167–173. doi: 10.1016/0014-5793(81)80197-4. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Swenson R. P., Howard J. B. Characterization of alkylamine-sensitive site in alpha 2-macroglobulin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4313–4316. doi: 10.1073/pnas.76.9.4313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Leuven F., Cassiman J. J., Van Den Berghe H. Demonstration of an alpha2-macroglobulin receptor in human fibroblasts, absent in tumor-derived cell lines. J Biol Chem. 1979 Jun 25;254(12):5155–5160. [PubMed] [Google Scholar]
  28. Van Leuven F., Cassiman J. J., Van den Berghe H. Functional modifications of alpha 2-macroglobulin by primary amines. I. Characterization of alpha 2 M after derivatization by methylamine and by factor XIII. J Biol Chem. 1981 Sep 10;256(17):9016–9022. [PubMed] [Google Scholar]
  29. 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]
  30. Wyckoff M., Rodbard D., Chrambach A. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers using multiphasic buffer systems: properties of the stack, valid Rf- measurement, and optimized procedure. Anal Biochem. 1977 Apr;78(2):459–482. doi: 10.1016/0003-2697(77)90107-5. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES