title: Mutational and immunochemical analysis of plasminogen activator inhibitor 1.
abstract: We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
authors.forename: H E
authors.initials: HE
authors.lastname: Shubeita
authors.forename: T L
authors.initials: TL
authors.lastname: Cottey
authors.forename: A E
authors.initials: AE
authors.lastname: Franke
authors.forename: R D
authors.initials: RD
authors.lastname: Gerard
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D015849:Plasminogen Inactivators / Q000276:immunology* / Q000378:metabolism*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors; D014361:Trypsin Inhibitors; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
chemical_list: D009838:Oligodeoxyribonucleotides; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D010196:Pancreatic Elastase; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator