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Title: Biochemical properties of recombinant single-chain urokinase-type plasminogen activator mutants with deletion of Asn2 through Phe157 and/or substitution of Cys279 with Ala
Authors: Lijnen, Roger ×
Li, X K
Nelles, Lucien
Hu, M H
Collen, Desire #
Issue Date: Apr-1992
Publisher: Published by Springer-Verlag on behalf of the Federation of European Biochemical Societies
Series Title: European Journal of Biochemistry vol:205 issue:2 pages:701-9
Abstract: The contribution of the NH2-terminal polypeptide chain and of the Cys148-Cys279 interchain disulphide bond to the enzyme activity of urokinase-type plasminogen activator (u-PA) was studied using site-specific mutagenesis. Recombinant single-chain u-PA (rscu-PA) variants were produced by transfecting Chinese hamster ovary cells with cDNA encoding des(Asn2-Phe157)rscu-PA (rscu-PA with deletion of Asn2-Phe157), [Ala279]rscu-PA (rscu-PA with Cys279----Ala mutation) or des(Asn2-Phe157)[Ala279]rscu-PA [des(Asn2-Phe157)rscu-PA with Cys279----Ala mutation]. Des(Asn2-Phe157)rscu-PA, [Ala279]rscu-PA and des(Asn2-Phe157)[Ala279]rscu-PA, purified from conditioned cell culture medium, were obtained as nearly homogeneous single-chain molecules with Mr approximately 30,000, 54,000 and 30,000, and specific fibrinolytic activities on fibrin plates of (mean +/- SD; n = 3) 860 +/- 150 IU/mg, 43.0 +/- 2.5 IU/micrograms and 240 +/- 20 IU/mg, respectively, compared to 69.0 +/- 4.3 IU/micrograms for wild-type rscu-PA obtained in the same expression system. The plasminogen activating potential in a buffer milieu of [Ala279]rscu-PA was somewhat lower than that of rscu-PA, but that of both deletion mutants was virtually abolished. In a human plasma milieu in vitro, consisting of a radiolabelled human plasma clot submerged in plasma, 50% clot lysis in 2 h required 6.5 micrograms/ml [Ala279]rscu-PA or 3.4 micrograms/ml rscu-PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 micrograms/ml. Treatment of [Ala279]rscu-PA or rscu-PA with plasmin resulted in quantitative conversion to two-chain molecules and was associated with an increase in specific amidolytic activity from about 600 IU/mg to 62.5 IU/micrograms for [Ala279]rscu-PA as compared to an increase from about 0.3 IU/micrograms to 75.0 IU/micrograms for rscu-PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2-Phe157)rscu-PA or des(Asn2-Phe157)[Ala279]rscu-PA with plasmin. The u-PA B-chain, isolated from plasmin-treated [Ala279]rscu-PA, had enzymic properties which were comparable to those of rtcu-PA, with respect to specific fibrinolytic activity, amidolytic activity, kinetics of plasminogen activation and clot-lysis activity in a human plasma milieu in vitro. Following bolus injection into hamsters, the plasma clearances were comparable (0.7-1.1 ml/min) for wild-type rscu-PA and for the three truncated rscu-PA mutants.(ABSTRACT TRUNCATED AT 400 WORDS)
ISSN: 0014-2956
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular and Vascular Biology
Vesalius Research Centre (-)
× corresponding author
# (joint) last author

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