MUTATION OF ARG154 TO GLY154 IN UROKINASE AUGMENTS ITS FIBRIN-SPECIFICITY

Rscu-PA and its mutant constructed by in vitro site specific mutagenes is of Arg154 in rscu-PA to Gly154 (mscu-PA) were both expressed in Esc herichia coli.. AR er in vitro denaturation and renaturation, the rscu -PA and mscu-PA were purified to homogeneity by Zn2+ selective precipi tation, anti-u-PA IgG-sepharose CL 4B affinity chromatography. after a ctivation by plasmin, the kinetic constants for the resultant mtcu-PA against synthetic substrate S2444 hydrolysis were found to be essentia lly identical to rtcu-PA, suggesting that no impairment had been exert ed on the catalytic active site of mtcu-PA. However, both I-125-fibrin plasma-clot lysis and fibrinogenolysis showed that mtcu-PA possessed a higher fibrinolytic activity but hardly any degradation of fibrinoge n in plasma compared to rtcu-PA and rscu-PA. It was concluded that the substitution of Arg 154 by Gly 154 in tcu-PA promoted the fibrin-spec ificity of urokinase.