Tropomyosin-enriched and alpha-actinin-enriched microfilaments isolated from chicken embryo fibroblasts by monoclonal antibodies.
Antitropomyosin and anti-alpha-actinin monoclonal antibodies have been used to isolate two classes of microfilaments, i.e., tropomyosin-enriched and alpha-actinin-enriched microfilaments, respectively, from cultured chicken embryo fibroblasts. Electron microscopic studies of the isolated tropomyosin-enriched microfilaments showed periodic localization of tropomyosin along the microfilaments, with a 35-nm repeat. On the contrary, the isolated alpha-actinin-enriched microfilaments showed no obvious periodicity. Many individual alpha-actinin-enriched microfilaments with length greater than 1 micron (ranging from 1 to 10 microns) were aggregated by anti-alpha-actinin monoclonal antibodies. Both of the isolated microfilaments had the ability to activate the Mg2+-ATPase activity of skeletal muscle myosin, although different extents of activation were observed. These two classes of microfilaments also differed in their protein composition. Molar ratios of major identifiable proteins in the isolated microfilaments were alpha-actinin(dimer):actin(monomer):tropomyosin(dimer) = less than 0.02:8.06:1.00 for tropomyosin-enriched microfilaments and 0.44:13.91:1.00 for alpha-actinin-enriched microfilaments. By two-dimensional gel analysis of the isolated microfilaments, we have found seven spots which possess typical tropomyosin properties including pI 4.5, immunological cross-reaction, lack of proline and tryptophan, and heat stability. Pulse-chase experiments suggested that the assembly of microfilament-associated proteins, at least for alpha-actinin and tropomyosins, was coordinately regulated by the assembly of actin into microfilaments.
D000185:Actinin / Q000302:isolation & purification* / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure*; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification*; D011485:Protein Binding; D014335:Tropomyosin / Q000302:isolation & purification* / Q000378:metabolism
D000199:Actins; D000911:Antibodies, Monoclonal; D009124:Muscle Proteins; D014335:Tropomyosin; D000185:Actinin