The tissue plasminogen activator finger domain confers fibrin-dependent enhancement of catalytic activity to single-chain urokinase-type plasminogen activator.
To determine whether the fibrin-binding domains of tissue plasminogen activator (tPA) can confer enhanced catalytic activity to single-chain urokinase-type plasminogen activator (scuPA), we constructed, expressed, and characterized the kinetics of five recombinant tPA/scuPA hybrid molecules. The hybrid molecules are: 1) tPA3-50 (tPA finger)/scuPA138-411, 2) tPA177-256 (tPA kringle2)/scuPA140-411 (scuPA catalytic), 3) tPA3-50/tPA177-256/scuPA140-411, 4) scuPA1-47 (scuPA growth factor)/tPA177-256/scuPA140-411, and 5) scuPA1-138 (scuPA growth factor and kringle)tPA127-256/scuPA139-411. The amidolytic activity of all hybrids was comparable, as were the kinetics for conversion from single-chain to two-chain plasminogen activator. We found that 1) the lag time prior to achieving maximal velocity among these hybrids varied, 2) hybrids 2, 3, 4, and 5 were 2-134-fold more potent (by kcat/Km) than hybrid 1, and 3) those hybrid proteins containing the tPA finger domain (hybrids 1 and 3) gave a 2-fold increase in catalytic efficiency in the presence of DESAFIB (reptilase-digested fibrinogen). These kinetic differences are likely mediated by changes in the tertiary structure of the scuPA catalytic domain resulting from interactions between catalytic and noncatalytic domains in the presence of fibrin.
Lubin
I M
IM
Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322.
Caban
R
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Runge
M S
MS
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D004247:DNA; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D004247:DNA; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator