Study on the activation of plasminogen by urokianse

Abstract

The activation of plasminogen to an active fibrinolytic enzyme plasmin, was studied with urokinase (EC 3.4.99.26). A crude form of urokinase obtained from human urine was purified partially by an ion-exchange column of ECTEOLA and further by an affinity column employing arginine as a ligand on sepharose 4B matrix. The specific activity of final enzyme preparation was about 301.4 units per mg protein based on the caseinolytic assay measuring the rate of casein hydrolysis by plasmin formed. The mode of the enzyme action seems to be similar to that of trypsin in the term of the esterase activity, but the enzyme is found to be very specific towards plasminogen as the substrate. Characteristic properties of the enzyme were studied and compared with that of other proteolytic enzymes of trypsin and plasmin. Therfore, it can be suggested that the nature of these enzymes may be different physiologically. Inhibition study of the enzyme was also carried out with a variable concentration of $Cl^{-}$ STI, 6-aminohexanoic acid and other substrate analogs. Studies on the mechanism of the enzyme action was carried out, in the manner of the effect of pH on kinetic constants and the inhibition by several functional group blocking agents, such as DIFP and diethylpyrocarbonate. These results indicate a possible involvement of hydroxyl group of serine and imidazole group of histidine residues at the active site of urokinase. The similarity of urokinase and other serine-proteases in mechanism is observed.