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 alpha(2)-antiplasmin, but not by alpha(1)-antitrypsin.