Amino-terminal fragment of urokinase-type plasminogen activator inhibits its plasminogen activation.
The amino terminal fragment (ATF, Ser(1)-Lys(135)) of urokinase-type plasminogen activator (uPA) containing an epidermal growth factor-like (EGF) and kringle domain is critically involved in some important functions of uPA, such as receptor binding and chemotactic activity. In this report, the effect of ATF on single-chain uPA (sc-uPA) induced plasminogen activation was investigated. It was shown that sc-uPA-induced activation of Glu-plasminogen or Lys-plasminogen was significantly inhibited in the presence of ATF. In addition, sc-uPA activation to two-chain uPA (tc-uPA) by Lys-plasmin and plasminogen activation to plasmin by tc-uPA were both found to be inhibited by ATF. The inhibition of these activations was significantly attenuated but not diminished when ATF was pretreated with immobilized carboxypeptidase B (CPB), indicating that the C-terminal Lys(135) as well as internal Lys/Arg residue binding was involved in the mechanism. Kinetic analysis showed that sc-uPA activation by Lys-plasmin competitively inhibited by ATF and CPB pretreated ATF (CPB-ATF) with an inhibitory constant (K(i)) of 3.8+/-0.31 and 12.4 +/- 1.8 microM, respectively. In contrast to sc-uPA-induced Glu- or Lys-plasminogen activation, sc-uPA-induced mini-plasminogen activation, sc-uPA activation by mini-plasmin and mini-plasminogen activation by tc-uPA were not affected by ATF. These findings suggested that the inhibitory effects of ATF on sc-uPA activation by Lys-plasmin and Glu- or Lys-plasminogen activation by tc-uPA were related to the binding of ATF (by its C-terminal Lys(135) and internal Lys/Arg residue) with the kringle 1-4 of plasmin and plasminogen, respectively.
Institute of Molecular Medicine, Nanjing University, Nanjing 210093, PR China.
Ziyong
Z
Sun
Pei-Xiang
PX
Zhang
Ping
P
Wang
Victor
V
Gurewich
Hai-Yan
HY
Shen
Jian-Ning
JN
Liu
D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005341:Fibrinolysin / Q000096:biosynthesis / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D018082:Kringles; D008322:Mammals / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D010958:Plasminogen / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; C033809:lys-plasmin; C018998:lysyl-plasminogen; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator