Covalent binding of proteinases in their reaction with alpha 2-macroglobulin.
Although it is known that most of the plasma proteinase inhibitors form complexes with proteinases that are not dissociated by SDS (sodium dodecyl sulphate), there has been disagreement as to whether this is true for alpha 2M (alpha 2-macroglobulin). We have examined the stability to SDS with reduction of complexes between alpha 2M and several 125I-labelled proteinases (trypsin, plasmin, leucocyte elastase, pancreatic elastase and papain) by gel electrophoresis. For each enzyme, some molecules were separated from the denatured alpha 2M chains, but amounts ranging from 8.3% (papain) to 61.2% (trypsin) were bound with a stability indicative of a covalent link. Proteolytic activity was essential for the covalent binding to occur, and the proteinase molecules became attached to the larger of the two proteolytic derivatives (apparent mol.wt. 111 000) of the alpha 2M subunit. We take this to mean that cleavage of the proteinase-susceptible site sometimes leads to covalent-bond formation between alpha 2M and proteinase. Whatever the nature of this bond, it does not involve the active site of the proteinase, as bound serine-proteinase molecules retain the ability to react with the active-site-directed reagent [3H]Dip-F (di-isopropyl phosphorofluoridate). Our conclusion is that the ability to form covalent links is not essential for the inhibitory capacity of alpha 2M. It may, however, help to stabilize the complexes against dissociation or proteolysis.
D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism*
D010446:Peptide Fragments; D000511:alpha-Macroglobulins; D012967:Sodium Dodecyl Sulfate; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase; D014357:Trypsin; D005341:Fibrinolysin; D010206:Papain