Mutational and immunochemical analysis of plasminogen activator inhibitor 1

Huda E. Shubeita, Theresa L. Cottey, Arthur E. Franke, Robert D. Gerard

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)18379-18385
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number30
StatePublished - Oct 25 1990

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Plasminogen Activators
Plasminogen Activator Inhibitor 1
Plasminogen Inactivators
Replication Protein C
Immunochemistry
Mutation
Pancreatic Elastase
Valine
Serine Proteases
Site-Directed Mutagenesis
Mutagenesis
Functional analysis
Thrombin
Trypsin
Serine
Lysine
Endothelial cells
Arginine
Endothelial Cells
Structural analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Shubeita, H. E., Cottey, T. L., Franke, A. E., & Gerard, R. D. (1990). Mutational and immunochemical analysis of plasminogen activator inhibitor 1. Journal of Biological Chemistry, 265(30), 18379-18385.

Mutational and immunochemical analysis of plasminogen activator inhibitor 1. / Shubeita, Huda E.; Cottey, Theresa L.; Franke, Arthur E.; Gerard, Robert D.

In: Journal of Biological Chemistry, Vol. 265, No. 30, 25.10.1990, p. 18379-18385.

Research output: Contribution to journalArticle

Shubeita, HE, Cottey, TL, Franke, AE & Gerard, RD 1990, 'Mutational and immunochemical analysis of plasminogen activator inhibitor 1', Journal of Biological Chemistry, vol. 265, no. 30, pp. 18379-18385.
Shubeita HE, Cottey TL, Franke AE, Gerard RD. Mutational and immunochemical analysis of plasminogen activator inhibitor 1. Journal of Biological Chemistry. 1990 Oct 25;265(30):18379-18385.
Shubeita, Huda E. ; Cottey, Theresa L. ; Franke, Arthur E. ; Gerard, Robert D. / Mutational and immunochemical analysis of plasminogen activator inhibitor 1. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 30. pp. 18379-18385.
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