On the conversion of high molecular weight urokinase to the low molecular weight form by plasmin

Abstract

The conversion of high molecular weight urokinase (HMW-UK) to low molecular weight urokinase (LMW-UK) by plasmin in vitro has been studied. The two forms of urokinase were separated by SDS-polyacrylamide gradient gel electrophoresis and active enzyme was extracted from 5 mm gel segments into isotonic saline and analyzed by the fibrin plate method. Electrophoretic separation of the two forms was complete, as indicated by comparison with the migration of purified standards and by the absence of lytic activity in extracts of intervening gel segments. HMW-UK was incubated with plasminogen and fibrinogen for various time intervals, after which enzymatic activity was inhibited with aprotinin and the samples subjected to electrophoresis. Conversion from HMW-UK to LMW-UK was apparent in the first sample at 2.5 minutes and continued for the 10 minute duration of the experiments. Similar experiments starting with LMW-UK showed no change in molecular weight. Incubation of HMW-UK without plasminogen resulted in no conversion to LMW-UK, indicating that plasmin was the active agent and that the reaction was not autocatalytic.