en
Scientific article
English

Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937

Published inThe Journal of biological chemistry, vol. 261, no. 24, p. 11207-11213
Publication date1986
Abstract

We report the production, purification, characterization, and partial amino acid sequence of a plasminogen inhibitor (PA-I). The starting material is culture fluid from phorbol myristate 13-acetate-treated U-937 cells and the isolation steps consist of preparative isoelectric focusing followed by affinity chromatography on Cibacron Blue-Sepharose. PA-I migrates as a closely spaced doublet of 47-kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forms covalent complexes with urokinase and two-chain tissue-type plasminogen activator, displaying second order rate constants of 0.9 X 10(6) M-1 s-1 and 0.2 X 10(6) M-1 s-1, respectively. Upon treatment with 1 M NH4OH, the covalent complexes were hydrolyzed, yielding a 35-kDa inhibitor fragment. A partial amino acid sequence of PA-I showed that it belongs to the antithrombin III family of inhibitors. PA-I is immunologically related to a PA-inhibitor from human placenta. mRNA from phorbol myristate 13-acetate-treated U-937 cells directed, in a rabbit reticulocyte derived cell-free system, the biosynthesis of only one 47-kDa protein that could be immunoprecipitated with anti-PA-I IgG, indicating that the two molecular forms of PA-I are the products of post-translational processing.

Keywords
  • Amino Acid Sequence
  • Cell Line
  • Chromatography, Affinity
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases
  • Glycoproteins/ isolation & purification
  • Humans
  • Kinetics
  • Lymphoma, Large B-Cell, Diffuse/ analysis
  • Mathematics
  • Placenta/analysis
  • Plasminogen Inactivators
  • Protease Inhibitors
  • Protein Biosynthesis
  • RNA, Messenger/analysis
  • Serine Endopeptidases
  • Tetradecanoylphorbol Acetate/pharmacology
  • Tissue Plasminogen Activator/metabolism
  • Urokinase-Type Plasminogen Activator/metabolism
Citation (ISO format)
KRUITHOF, Egbert et al. Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937. In: The Journal of biological chemistry, 1986, vol. 261, n° 24, p. 11207–11213.
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ISSN of the journal0021-9258
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