Abstract
A procedure was developed for the purification of a tissue plasminogen activator from hog kidney. It involves six consecutive steps : (1) extraction of the tissue plasminogen activator from acetone-dried hog kidney with 0.3M potassium acetate buffer, pH 4.2 ; (2) ammonium sulfate precipitation ; (3) hydrophobic chromatography on n-butyl-Sepharose ; (4) affinity chromatography on concanavalin A-Sepharose ; (5) gel filtration on hydrophilic vinylmonomer (Toyopearl HW-55) ; (6) affinity chromatography on fibrin-Sepharose. The purified tissue plasminogen activator had an activity of 13000 IU per mg of protein as assessed by plasminogen activation and had no direct fibrinolytic activity. The apparent molecular weight of the purified tissue plasminogen activator was 60000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analyses of the purified plasminogen activator preparation showed it to contain 54.0 μg of sialic acid and 175.0μg of hexose per mg of protein. As assessed by plasmin-catalyzed hydrolysis of D-Val-Leu-Lys-pNA, the functional activity of the purified tissue plasmigogen activator was markedly stimulated by addition of fibrin, whereas the interaction with fibrin had a slightly stimulating effect on the activity of human urokinase. Treatment with concanavalin A or glycosidases resulted in 75% or 70% loss of activity of tissue plasminogen activator, respectively, but had no effect on human urokinase.
