Inhibition of actin filament movement by monoclonal antibodies against the motor domain of myosin.
Conformational transitions in defined regions of the motor domain of skeletal muscle myosin involved in ATP hydrolysis, actin binding and motility were probed with monoclonal antibodies. Competition binding assays demonstrate that three different monoclonal antibodies react with spatially related sites on the myosin heavy chain. One recognizes a sequential epitope between residues 65 and 80 and has no effect on actin filament movement in an in vitro motility assay despite tight binding to myosin and acto-S1. The other two monoclonal antibodies react with sequential epitopes between residues 29 and 60 and both inhibit actin filament movement. A fourth monoclonal antibody reacts with the N-terminus of the heavy chain (residues 1-12) at a spatially distinct site on the myosin head and also inhibits actin filament movement. These four monoclonal antibodies have been mapped by immunoelectron microscopy to the large, actin binding region of the myosin head; however, the antibody binding sites remain accessible on rigor complexes of acto-S1. Thus, this group of monoclonal antibodies identify sequential epitopes in a mobile segment of the myosin heavy chain. In addition, two conformation-sensitive monoclonal antibodies are described that are affected by ATP and actin binding to myosin S1, and display distinct and marked inhibitory effects on actin filament movement. In contrast, an anti-light chain monoclonal antibody that binds near the myosin head-rod junction has little effect on the number and velocity of moving actin filaments. These results identify mobile regions on the myosin head that are perturbed by antibody binding and that may be linked to force production and motion.
Winkelmann
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DA
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ 08854.
Kinose
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F
Chung
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D000199:Actins / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D002645:Chickens; D000939:Epitopes / Q000276:immunology; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008969:Molecular Sequence Data; D009038:Motion; D009218:Myosins / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014743:Videotape Recording
D000199:Actins; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D009218:Myosins