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Characterisation of a mutant of recombinant human single chain urokinase-type plasminogen activator (scu-PA), obtained by substitution of arginine 156 and lysine 158 with threonine

par Lijnen, Henri Roger;Collen, Désiré;Gheysen, Dirk;de Foresta, Françoise;Pierard, Laurent ;Jacobs, Paul ;Bollen, Alex
Référence Fibrinolysis and Proteolysis, 2, 2, page (85-93)
Publication Publié, 1988

Article révisé par les pairs

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Résumé :

A mutant of recombinant single chain urokinase-type plasminogen activator (rscu-PA) was constructed by site specific mutagenesis of Arg156 and Lys158 to Thr (rscu-PA-Thr156,158) and expressed in hamster kidney cells using a bovine papilloma virus (BPV) derived vector. The mutant was purified to homogeneity by chromatography on Zinc chelate-Sepharose and immunoadsorption on an insolubilised monoclonal antibody raised against natural u-PA. The specific activity of the mutant scu-PA on fibrin plates was very low (350 IU/mg) compared to that of rscu-PA (63 000 IU/mg). The mutant, in contrast to rscu-PA, was not converted to a two-chain molecule by plasmin or thrombin and did not cause lysis of a 125I-fibrin labelled plasma clot immersed in citrated plasma. However, in a purified system, rscu-PA-Thrl56,158 activated plasminogen following Michaelis-Menten kinetics, with a much lower affinity (Km = 44 μM) but with a comparable catalytic rate constant (k2 = 0.0076 s-1) as compared to rscu-PA (Km = 0.67 μM, k2 = 0.0022 s-1). These findings indicate that this mutant in which the plasmin and thrombin cleavage sites are eliminated, can convert plasminogen to plasmin without requiring prior conversion to a two chain molecule but its catalytic efficiency is about 20 fold lower than that of rscu-PA. © 1988.