Inactivation of the intrinsic activity of pro-urokinase by diisopropyl fluorophosphate is reversible.
Single chain urokinase-type plasminogen activator or pro-urokinase (pro-UK) has been reported to have a significant intrinsic amidolytic and plasminogen activator activity, estimated to be about 0.2-0.6% that of two-chain urokinase (UK). However, it has also been suggested that this reported activity is related entirely to trace UK contaminants generated during the analytic procedures. In an attempt to resolve this controversy, it was decided to measure the incorporation of diisopropyl fluorophosphate (DFP) by pro-UK and UK. Surprisingly, it was found that although > 98% of the apparent intrinsic activity of pro-UK was inhibited by 5 mM DFP, > 97% of this activity was recoverable after exhaustive dialysis of the preparation. This finding could not be explained by UK generation, which was excluded. Instead, the findings indicated that DFP inhibition of pro-UK, in contrast to UK and other serine proteases, was largely reversible. The reaction rate of the reversible inhibition was significantly slower than that of irreversible inhibition by DFP. When the hydrolysis of DFP (2 mM) during incubation (37 degrees C) with or without pro-UK (20 microM) was compared, a > 5-fold acceleration of DFP hydrolysis in the presence of pro-UK was found, whereas little loss of DFP occurred in the presence of UK (20 microM), consistent with 1:1 stoichiometry. This suggested that pro-UK acted as a slow DFPase in the reaction, a finding consistent with a reversible DFP-enzyme reaction. It was concluded that pro-UK has a distinct and measurable intrinsic catalytic activity, which is qualitatively unique and thereby distinguishable from that of UK as well as other serine proteases.
Liu
J N
JN
Vascular Research Laboratory, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA.
Gurewich
V
V
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D003956:Dialysis; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors*; D006868:Hydrolysis; D007531:Isoflurophate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*
D000577:Amides; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D007531:Isoflurophate; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase