Three high molecular weight protease inhibitors of rat plasma. Reactions with trypsin.
We have compared the reactions of trypsin with human alpha 2-macroglobulin (alpha 2M), and three rat plasma protease inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2M. All four of these proteins appear to contain reactive thiol esters. The electrophoretic mobility in agarose gels of human and rat alpha 2M is increased by 1 mol of trypsin, while the mobility of alpha 1M and alpha 1I3 is decreased. Treatment with methylamine causes similar mobility changes, except in the case of rat alpha 2M. Titration of human and rat macroglobulins by repeated small additions of trypsin and by assay of liberated SH groups or enhanced ligand fluorescence revealed a stoichiometry of about 1 mol of trypsin/mol of inhibitor. In contrast, addition of macroglobulin to a fixed amount of trypsin and detection of residual amidase or protease activity revealed a stoichiometry of about 2 mol of trypsin for 1 mol of human alpha 2M, about 1.4 mol for rat alpha 1M, and about 1 mol for rat alpha 2M. One mol of trypsin reacted with 2 or more mol of alpha 1I3 by the criteria of SH groups liberated or protease inhibition. Methylamine-treated rat alpha 2M binds a significant amount of trypsin releasing about 2 mol of SH. Radioactive beta-trypsin was covalently bound to subunits of the purified plasma inhibitors. The Mr of the labeled products with rat and human alpha 2M had molecular weights which suggested trypsin was bound to intact as well as cleaved subunit chains and also to multiple chains via cross-linking. Rat alpha 1M also produced a product which may be an intact subunit alpha chain plus trypsin. Greater than 80% of the trypsin was bound covalently to these inhibitors at low molar ratios.
D000209:Acute-Phase Proteins; D000818:Animals; D001665:Binding Sites; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000097:blood* / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D013438:Sulfhydryl Compounds; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
D000209:Acute-Phase Proteins; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D014361:Trypsin Inhibitors; C017894:alpha 1-inhibitor 3; D000511:alpha-Macroglobulins; D014357:Trypsin