Activation of Plasminogen by Single - Chain Urokinase or by Two - Chain Urokinase - A Demonstration

Single-chain urokinase (SC-UK) has an intrinsic amidolytic activity. as measured with synthetic substrate (Kabi 52444; pyro-GIu-GIy-Arg-pNitroanalide). which was found to be 0.1 % to 0.2% that of its plasmin-activated derivative, two-chain UK (TC-UK). A study of the reaction of SC-UK with plasminogen is complicated by the effect of the reaction product, plasmin, on both reactants. The resultant generation of TC-UK and Lys-plasminogen produces secondary reactions which greatly augment plasminogen activation. To confine enzymatic activity to the primary reaction, after pretreatment to eliminate trace TC-UK contaminants. SC-UK was incubated with Gluor Lysplasminogen in the presence of aprotinin (500 KIU/mL) to inhibit generated plasmin and dansyl-glutamyl-glycyl-arginyl-chloromethylketone (20 zmoI/L). which irreversibly inhibited TC-UK but not SC-UK. Analysis by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a plasminogen-activating activity for SC-UK that was -0.4% that of TC-UK. Both SC-UK and TC-UK preferentially activated Lys-plasminogen over Gluplasminogen. Similarly. Glu-plasminogen activation was augmented by lysine or soluble fibrin. The ratio of the reaction rates of SC-UK and TC-UK were comparable for Gluand Lys-plasminogen. It is concluded that there is a major difference in the catalytic activities of SC-UK and TC-UK against plasminogen that is comparable to that against synthetic substrate. S 1987 by Grune & Stratton, Inc.