Purification and characterization of native bovine milk plasminogen activators
Abstract
The breakdown of milk proteins by proteases affects the quality of dairy products. The proteolysis results in changes in bovine milk viscosity, solubility, cheesemaking characteristics, cheese ripening and age gelation of UHT milk. The milk plasmin system, responsible for the proteolysis, includes plasmin, plasminogen, plasmin inhibitor, plasminogen activator and plasminogen activator inhibitor. Plasmin, which actually breaks down the milk protein, exists mainly in milk in its inactive form, plasminogen. Activation of plasminogen to plasmin is catalyzed by plasminogen activators. Plasminogen activators were partially purified by 2204-fold from fresh bovine skim milk, which contained 340 mU/L plasminogen activator activity. Partially purified plasminogen activators, visualized by casein-plasminogen SDS-PAGE, had molecular weights of 93.0, 57.4, 41.6, 35.4, and 27.1 kDa. Most or all of the plasminogen activators in bovine milk were urokinase-type, as opposed to tissue-type. The plasminogen activators were inhibited by the serine proteinase inhibitors TLCK (1-chloro-3-tosylamido-7-amino-2-heptanone), PMSF (phenylmethylsulfonyl fluoride), and TPCK (L-1-tosylamido-2-phenylethyl chloromethyl ketone), PAI-1 (endothelial cell-type PA inhibitor), plasminogen activator inhibitor from erythrina seed, and alpha$\sb2$-antiplasmin, but not inhibited by alpha$\sb1$-antitrypsin. Kinetics studies of the heat-inactivation of plasminogen activators purified indicated that native plasminogen activators in bovine milk are not affected by pasteurization processes, and largely are not inactivated by UHT processing conditions used in the dairy industry. Plasmin was purified 41,709-fold from bovine milk sodium caseinate, which contained 30.7 mU/g plasmin activity. The plasmin purified from sodium caseinate was identical with the plasmin from bovine blood. An additional protease found to be present in the sodium caseinate was proven not to be thrombin.
Degree
Ph.D.
Advisors
Nielsen, Purdue University.
Subject Area
Food science
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.