title: Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3.
abstract: The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.
authors: Duke University Medical Center, Durham, North Carolina 27710.
authors: J J
authors: JJ
authors: Enghild
authors: G
authors: G
authors: Salvesen
authors: I B
authors: IB
authors: Thøgersen
authors: S V
authors: SV
authors: Pizzo
mesh_terms: D000209:Acute-Phase Proteins; D000818:Animals; D001665:Binding Sites; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D006868:Hydrolysis; D007106:Immune Sera; D007704:Kininogens / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010206:Papain / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D012697:Serine Endopeptidases; D000511:alpha-Macroglobulins / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000209:Acute-Phase Proteins; D007106:Immune Sera; D007704:Kininogens; D046911:Macromolecular Substances; D011480:Protease Inhibitors; C017894:alpha 1-inhibitor 3; D000511:alpha-Macroglobulins; D002403:Cathepsins; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C534094:CTSG protein, human; D056649:Cathepsin G; C534097:Ctsg protein, rat; D010196:Pancreatic Elastase; D010206:Papain; D008239:Lysine