title: Characterization of recombinant human alpha 2-antiplasmin and of mutants obtained by site-directed mutagenesis of the reactive site.
abstract: Human alpha 2-antiplasmin (alpha 2AP) has been expressed in Chinese hamster ovary cells and purified from conditioned media. The recombinant protein (r alpha 2AP) is immunologically identical with natural alpha 2AP and indistinguishable with respect to plasmin(ogen) binding properties. Second-order rate constants (k1) for the interaction of alpha 2AP and r alpha 2AP with plasmin are both (1-2) X 10(7) M-1 s-1. In order to examine the effects of alterations within the reactive site of alpha 2AP, deletions of the P1 residue Arg-364 (r alpha 2AP-delta Arg364) or the P'1 residue Met-365 (r alpha 2AP-delta Met365) were introduced by in vitro site-directed mutagenesis. r alpha 2AP-delta Met365 completely retains its ability to inhibit both plasmin and trypsin, indicating that alpha 2AP has no absolute requirement for Met in the P'1 position. Unexpectedly, no increase in antithrombin activity was observed. r alpha 2AP-delta Arg364 has lost the ability to inhibit plasmin, trypsin, and thrombin, but unlike the wild-type protein, this variant is an effective elastase inhibitor (k1 = 1.5 X 10(5) M-1 s-1).
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D000979:alpha-2-Antiplasmin / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin