Studies on the structure and conformation of brush border myosin using monoclonal antibodies.
We have produced and characterised five monoclonal antibodies against myosin isolated from chicken intestinal epithelial brush border cells. The binding sites of the antibodies on the rod portion of brush border myosin were localised using rotary shadowing/electron microscopy of myosin-antibody complexes. Two antibodies were shown to bind to the tip of the myosin tail, two antibodies to sites about two thirds down the length of the rod, and one antibody about one third down the length of the rod. Brush border myosin was digested with papain, trypsin and alpha-chymotrypsin, and they myosin fragments obtained were analysed by western blots with the monoclonal antibodies and polyclonal antiserum, and by gel overlay with 125I-labelled light chains. Using this approach we were able to identify and map the protease cleavage sites and thus characterise the proteolytic substructure of brush border myosin. Solid-phase assays, western blots and immunofluorescence were used to study the cross-reactivity of these monoclonal antibodies against a variety of myosins from different species and cell types, to assess the immunological relatedness between brush border myosin and homologous molecules present in different tissues and species. Finally, we used a competitive solid-phase assay to measure the 'relative affinities' of the antibodies towards the three possible conformational states of brush border myosin, i.e. filament, extended monomer and folded monomer.
Citi
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Kendrick-Jones
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001666:Binding Sites, Antibody; D055598:Chemical Phenomena; D002621:Chemistry; D002645:Chickens; D003429:Cross Reactions; D007120:Immunochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008871:Microvilli / Q000032:analysis*; D009218:Myosins / Q000032:analysis* / Q000276:immunology; D010447:Peptide Hydrolases; D011487:Protein Conformation; D051381:Rats
D000911:Antibodies, Monoclonal; D010447:Peptide Hydrolases; D009218:Myosins