A study of proteases and protease-inhibitor complexes in biological fluids.
We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to alpha2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to alpha2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 mul of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely.
D000362:Agar; D000818:Animals; D001826:Body Fluids / Q000201:enzymology*; D002460:Cell Line; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008722:Methods; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis* / Q000378:metabolism; D010960:Plasminogen Activators / Q000032:analysis; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins / Q000378:metabolism
D003470:Culture Media; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D000362:Agar; D010447:Peptide Hydrolases; D010960:Plasminogen Activators