Single-chain urokinase-type plasminogen activator does not possess measurable intrinsic amidolytic or plasminogen activator activities.
The question whether single-chain urokinase-type plasminogen activator (Sc-uPA) possesses an enzymatic activity has been a subject of intense investigation for a number of years but still remains unresolved. Recent studies from several laboratories suggest that Sc-uPA or its plasmin-resistant mutants obtained by site-directed mutagenesis possess significant, albeit low, amidolytic and plasminogen activator activities, ranging from 0.1% to 1% of that observed for two-chain urokinase (Tc-uPA). In an effort to characterize these putative intrinsic activities, Sc-uPA was repeatedly treated with dansyl-Glu-Gly-Arg chloromethyl ketone (dansyl-EGRck) or diisopropyl fluorophosphate (DFP) (0.1-0.25 mM added thrice over a period of 24 h at 0 degrees C). This treatment exhaustively inactivated the Tc-uPA contaminant but did not affect Sc-uPA, as evidenced by the lack of significant incorporation of radiolabeled inhibitor in Sc-uPA and full activation of the inhibitor-treated Sc-uPA by plasmin. Assayed in the presence of excess DFP or dansyl-EGRck to ensure trapping of any Tc-uPA generated in the assay mixture, Sc-uPA (84 micrograms/mL, 10,500 latent units/mL) did not elicit any detectable cleavage of the chromogenic substrate S-2444 (detection limit 0.1 unit of Tc-uPA/mL). However, if the Tc-uPA inhibitors were removed prior to assay, a trace amount of amidolytic activity invariably reappeared in the Sc-uPA preparation. Incorporation experiments with [3H]DFP suggested that the appearance of this amidolytic activity was due to formation of Tc-uPA. Plasminogen activator assay of DFP- and dansyl-EGRck-treated Sc-uPA (0.45-2.25 microM), performed in the presence of these inhibitors and Trasylol (10 microM) to ensure entrapment of any Tc-uPA or plasmin generated in the reaction mixture, showed no significant cleavage of 125I-labeled plasminogen (detection limit 0.1 nM). However, if dansyl-EGRck and DFP were removed from the inhibitor-treated Sc-uPA and the assay was performed in the presence of Trasylol alone, there was significant cleavage of 125I-plasminogen due to contamination by Tc-uPA. Fibrin, a positive effector of plasminogen activation by Tc-uPA or Sc-uPA preparations in the absence of DFP and dansyl-EGRck, did not promote cleavage of plasminogen or S-2444 by Sc-uPA in the presence of the Tc-uPA inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)
Husain
S S
SS
Department of Anesthesia, Massachusetts General Hospital, Boston 02114.
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D006224:Cricetinae; D003412:Cricetulus; D003619:Dansyl Compounds; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005337:Fibrin / Q000494:pharmacology; D005341:Fibrinolysin / Q000494:pharmacology; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007531:Isoflurophate / Q000494:pharmacology; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism; D010053:Ovary / Q000187:drug effects / Q000201:enzymology*; D010960:Plasminogen Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D004792:Enzyme Precursors; D005343:Fibrinolytic Agents; D009842:Oligopeptides; D011994:Recombinant Proteins; D007531:Isoflurophate; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; C028190:5-oxo-prolyl-glycyl-arginine-4-nitroanilide; D005337:Fibrin; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator