title: Construction of Urokinase Mutant Glu154-mtcu-PA and Characterization of Its Properties.
abstract: The recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a mutant constructed by in vitro site-specific mutagenesis of Argl54 in rscu-PA to Glul54 (Glul54-mscu-PA) were both expressed in E. coli. The expressed products were both purified to homogeneity by in vitro denaturation and renaturation, followed by Zn(2+) selective precipitation and immuno-affinity chromatography. The plasmin sensitivity assay indicated that the activation of this single chain Glul54-mscu-PA by plasmin was essentially identical to that of rscu-PA. After activation by plasmin, the kinetic constants against synthetic substrate S2444 of the resulted two chain form of Glul54-mscu-PA (Glul54-mtcu-PA) and that of rscu-PA (rtcu-PA) were 87 &mgr;M and 80 &mgr;M, respectively, which indicated that the catalytic active site of the Glul54-mtcu-PA was not changed by the mutation. Whereas, both (125)I-fibrin plasma-clot lysis and fibrinogenolysis in plasma showed that the Glul54-mtcu-PA possessed a better affinity and selectivity for fibrin than rtcu-PA, even better than rscu-PA.
authors.affiliation: Department of Biochemistry and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China.
authors.forename: Gui-Hong
authors.initials: GH
authors.lastname: Peng
authors.forename: Zhong
authors.initials: Z
authors.lastname: Ma
authors.forename: Yu-Ming
authors.initials: YM
authors.lastname: Xue
authors.forename: Yu-Hong
authors.initials: YH
authors.lastname: Chen
authors.forename: De-Xu
authors.initials: DX
authors.lastname: Zhu