Mutation of Arg154 to Gly154 in urokinase augments its fibrin-specificity.
Rscu-PA and its mutant constructed by in vitro site specific mutagenesis of Arg154 in rscu-PA to Gly154 (mscu-PA) were both expressed in Escherichia coli. After in vitro denaturation and renaturation, the rscu-PA and mscu-PA were purified to homogeneity by Zn2+ selective precipitation, anti-u-PA IgG-sepharose CL 4B affinity chromatography. After activation by plasmin, the kinetic constants for the resultant mtcu-PA against synthetic substrate S2444 hydrolysis were found to be essentially identical to rtcu-PA, suggesting that no impairment had been exerted on the catalytic active site of mtcu-PA. However, both 125I-fibrin plasma-clot lysis and fibrinogenolysis showed that mtcu-PA possessed a higher fibrinolytic activity but hardly any degradation of fibrinogen in plasma compared to rtcu-PA and rscu-PA. It was concluded that the substitution of Arg154 by Gly154 in tcu-PA promoted the fibrin-specificity of urokinase.
Peng
G
G
Department of Biochemistry, Nanjing University, P.R.China.
Ma
Z
Z
Kuai
L
L
Zhu
D
D
D001120:Arginine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005337:Fibrin / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D005337:Fibrin; D001120:Arginine; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D005998:Glycine