Evidence for the tight binding of human mucus proteinase inhibitor to highly glycosylated macromolecules in sputum.
Two extraction procedures of non-purulent sputum for the isolation of human mucus proteinase inhibitor (MPI) in its free and bound forms have been assayed. The dissociating procedure involved sputum homogenization in 1M NaCl and 4% (w/v) trichloroacetic treatment. When the soluble material was applied to a CM-Trisacryl column, a non-negligible, MPI-related inhibitory activity was recovered with the highly glycosylated constituents not retained on the column; the amount of MPI released in a free form was retained and eluted from the column according to the basic character of this inhibitor. The non-dissociating procedure consisted in a high water dilution (1:12) of sputum, known to bring into solution the macromolecular, fibrillar constituents, which was followed by ultrafiltration on selected Mr cut-off membranes. All the inhibitory activity was recovered with the high Mr (greater than 100,000) fraction which was shown on SDS-PAGE to be essentially composed of strongly glycosylated material; on electrophoretic analysis under non-reducing conditions, the MPI activity was visualized as three bands which corresponded to the inhibitor released from this high Mr fraction in the presence of SDS. As mucin-type molecules are the major, highly glycosylated constituents of bronchial secretions, it is suggested that they are responsible for the entrapping of MPI within their macromolecular network; it would appear that, as well as for lysozyme, electrostatic interactions occur between the acid charges of mucins and the basic charges of MPI. The possible in vivo consequences of these interactions on MPI activity are discussed.
Unité INSERM No. 16, Lille, France.
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I
Van-Seuningen
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M
Davril
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A
Hayem
D001980:Bronchi / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D009093:Mucus / Q000032:analysis*; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding; D013183:Sputum / Q000378:metabolism*; D014462:Ultrafiltration
D006023:Glycoproteins; D011480:Protease Inhibitors