ISOLATION AND CHARACTERIZATION OF PLASMINOGEN AND PLASMIN FROM BOVINE-MILK

A procedure was developed for the isolation of plasminogen and plasmin from bovine milk. Pure undegraded plasminogen with N-terminal amino a cid sequence and mobility in SDS-PAGE similar to plasminogen isolated from bovine blood was obtained. Additionally, the preparation containe d the proteolytically modified midi-plasmin, consisting of kringle 4, 5 and the light chain with an apparent molecular weight of 50 kDa in u nreduced SDS-PAGE, and resulting in two bands of 30 and 26 kDa after r eduction. Some 15% of the amino acid sequence of plasminogen/midi-plas min isolated from milk was determined. The partial amino acid sequence was identical to that previously reported for plasminogen isolated fr om bovine blood (J. Schaller et al., (1985). Eur. J. Biochem., 149, 26 7-78), and to the bovine liver plasminogen cDNA sequence (L. Berglund et al., (1995). Int. Dairy J., 5, 593-603). Enzyme-linked immunosorben t assay (ELISA) and Western blotting experiments revealed that immunor eactive plasminogen was associated with acid-precipitated casein, renn et-coagulated casein and casein micelles. Some was found in acid whey and to a lesser extent in rennet whey. The amount of plasminogen assoc iated with the milk fat globule membrane was very low and reflected th e presence of casein. By Western blotting, immunoreactive plasminogen was found in zones with apparent molecular weights of 85,000 and 50 kD a in unreduced gels. These bands may represent two plasminogen forms ( 85 and 80 kDa) and midi-plasmin/midi-plasminogen (50 kDa). The total c oncentration of plasminogen in bovine milk was estimated to 1.5 mu g/m L.