Plasminogen activation with single-chain urokinase-type plasminogen activator (scu-PA). Studies with active site mutagenized plasminogen (Ser740----Ala) and plasmin-resistant scu-PA (Lys158----Glu)

J Biol Chem. 1990 Mar 25;265(9):5232-6.


The mechanism of the activation of plasminogen by single-chain urokinase-type plasminogen activator (single-chain u-PA, scu-PA) was studied using rscu-PA-Glu158, a recombinant plasmin-resistant mutant of human scu-PA obtained by site-specific mutagenesis of Lys158 to Glu, and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active-site Ser740 to Ala. Conversion of 125I-labeled single-chain plasminogen to two-chain plasmin was quantitated on reduced sodium dodecyl sulfate-gel electrophoresis combined with autoradiography and radioisotope counting of gels bands. The efficiencies of both rscu-PA-Glu158 and rscu-PA for the activation of rPlg-Ala740 and of natural plasminogen were comparable and were 250-500-fold lower than that of recombinant two-chain u-PA (rtcu-PA) for rscu-PA-Glu158 and 100-200-fold lower for rscu-PA. Pretreatment of rscu-PA-Glu158 or rscu-PA with excess alpha 2-antiplasmin, which efficiently neutralizes all contaminating rtcu-PA, did not significantly reduce the catalytic efficiency of these single-chain moieties, indicating that they have a low but significant intrinsic plasminogen activating potential. The low intrinsic catalytic efficiency of rscu-PA for the conversion of plasminogen to plasmin may be sufficient to generate trace amounts of plasmin, which may regulate plasminogen activation by converting poorly active rscu-PA to very active rtcu-PA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine*
  • Enzyme Activation
  • Enzyme Precursors / metabolism*
  • Escherichia coli / genetics
  • Fibrinolysin / metabolism*
  • Glutamates*
  • Glutamic Acid
  • Humans
  • Kinetics
  • Lysine*
  • Mutation
  • Plasminogen / genetics
  • Plasminogen / metabolism*
  • Plasminogen Activators / metabolism*
  • Recombinant Proteins / metabolism
  • Serine*
  • Urokinase-Type Plasminogen Activator / metabolism*


  • Enzyme Precursors
  • Glutamates
  • Recombinant Proteins
  • Glutamic Acid
  • Serine
  • Plasminogen
  • Plasminogen Activators
  • Fibrinolysin
  • Urokinase-Type Plasminogen Activator
  • Lysine
  • Alanine