Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937.
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.
D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D016403:Lymphoma, Large B-Cell, Diffuse / Q000032:analysis*; D008433:Mathematics; D010920:Placenta / Q000032:analysis; D015849:Plasminogen Inactivators; D011480:Protease Inhibitors; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D012697:Serine Endopeptidases; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D006023:Glycoproteins; D015849:Plasminogen Inactivators; D011480:Protease Inhibitors; D012333:RNA, Messenger; D010450:Endopeptidases; D012697:Serine Endopeptidases; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate