title: Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade fibronectin: their increase after sodium butyrate treatment of the cells.
abstract: Mouse bone marrow-derived mast cells, differentiated in vitro with concanavalin A splenocyte-conditioned medium and sensitized with monoclonal IgE, release neutral serine proteases after activation with specific antigen. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the supernatants from immunologically activated mast cells revealed the presence of four prominent proteins of 27,000, 29,000, 30,000 and 31,000 m.w. When the supernatants and sonicated residual cells from antigen-challenged or nonactivated IgE-sensitized mast cells were incubated with [3H]diisopropylfluorophosphate ([3H]DFP) and the proteins were subjected to SDS-PAGE followed by autoradiography, proteins of 27,000 to 31,000 m.w. were labeled with [3H]DFP. The antigen-dependent release of labeled proteins was accompanied by a corresponding depletion of similarly sized [3H]DFP-labeled proteins from these cell pellets relative to unactivated cells. The SDS gels were also stained with Coomassie Blue and were sectioned to separate the individual proteins for measurement of their incorporated radioactivity; the net percent antigen-dependent release of all four [3H]DFP-labeled proteins ranged from 64 to 68% and was comparable to that of the secretory granule markers, beta-hexosaminidase and histamine. That the [3H]DFP-labeled proteins were derived from the secretory granules of the cells was supported by studies in which mast cells were cultured for 4 days in the presence of 1 mM sodium butyrate. This treatment produced a differential increase in their cellular content of histamine (10-fold), [3H]DFP binding proteins (two- to fourfold), and beta-hexosaminidase (minimally), while the net percent antigen-dependent release of each of these constituents was unchanged. After sensitization and antigen activation, the net percent release of histamine, beta-hexosaminidase, and the four [3H]DFP-labeled proteins was 51, 59, and 53 to 61%, respectively, for sodium butyrate-treated cells, and 53, 60, and 64 to 68%, respectively, for cells not exposed to sodium butyrate. Human plasma fibronectin was used as a substrate to demonstrate that the exocytosed proteins possessed proteolytic activity. As assessed by optical density scanning of stained SDS-PAGE gels of the substrate, the proteases present in the supernatants of antigen-activated cells, but not of sensitized unchallenged cells, rapidly degraded native fibronectin at pH 7.0. This degradation was prevented by pretreatment of the exocytosed proteins from immunologically activated cells for 90 min at 37 degrees C with 2 mM DFP.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D003594:Cytoplasmic Granules / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism* / Q000502:physiology; D005089:Exocytosis; D005353:Fibronectins / Q000378:metabolism*; D006636:Histamine Release / Q000187:drug effects; D007073:Immunoglobulin E / Q000502:physiology; D007531:Isoflurophate / Q000378:metabolism; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D011485:Protein Binding / Q000187:drug effects; D012697:Serine Endopeptidases
chemical_list: D002087:Butyrates; D005353:Fibronectins; D020148:Butyric Acid; D007531:Isoflurophate; D007073:Immunoglobulin E; D010450:Endopeptidases; D012697:Serine Endopeptidases