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

Schleuning, W. D.
Mattaliano, R. J.
Bachmann, F.
Published in The Journal of biological chemistry. 1986, vol. 261, no. 24, p. 11207-11213
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 SequenceCell LineChromatography, AffinityElectrophoresis, Polyacrylamide GelEndopeptidasesGlycoproteins/ isolation & purificationHumansKineticsLymphoma, Large B-Cell, Diffuse/ analysisMathematicsPlacenta/analysisPlasminogen InactivatorsProtease InhibitorsProtein BiosynthesisRNA, Messenger/analysisSerine EndopeptidasesTetradecanoylphorbol Acetate/pharmacologyTissue Plasminogen Activator/metabolismUrokinase-Type Plasminogen Activator/metabolism
PMID: 3090045
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Other version: http://www.jbc.org/content/261/24/11207.full.pdf
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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. https://archive-ouverte.unige.ch/unige:8839

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Deposited on : 2010-07-12

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