Isolation and Characterization of Native Bovine Milk Plasminogen Activators

Plasminogen activators were partially purified from fresh bovine skim milk by treatments with sulfuric acid, ammonium sulfate, and dimethylformamide, followed by Zn-chelating chromatography, resulting in a purification factor of 2204-fold for skim milk, which contained 340 mU/L of plasminogen activator activity as measured by a colorimetric assay. Further purification with plasminogen activator inhibitor affinity chromatography gave purification factors of about 11,000-fold, but plasminogen activators in this fraction were not stable. The plasminogen activators obtained from Zn-chelating chromatography were characterized for molecular mass, urokinase-type versus tissue-type, and susceptibility to protease inhibitors. Five bands with plasminogen activator activity were detected by casein-plasminogen SDS-PAGE with molecular mass of approximately 93, 57, 42, 35, and 27 kDa. Most or all of the plasminogen activators in bovine milk were urokinase-type; the activity of the bovine milk plasminogen activators was not enhanced by the presence of fibrin. The immunological dissimilarity between bovine milk plasminogen activators and human urokinase-type plasminogen activator and human tissue-type plasminogen activator was shown by antibody quenching tests. Bovine milk plasminogen activators were inhibited by certain serine proteinase inhibitors, endothelial cell-type plasminogen activator inhibitor, plasminogen activator inhibitor from erythrina seed, and α 2 -antiplasmin, but not by α 1 -antitrypsin.