Binding of myosin subfragment 1 to glycerinated insect flight muscle in the rigor state.
The binding of rabbit muscle myosin subfragment 1 (S1) to glycerinated insect flight muscle fibers has been studied by low-angle x-ray diffraction, quantitative sodium dodecyl sulfate gel electrophoresis, quantitative interference microscopy, and electron microscopy. Changes induced in the rigor x-ray diffraction pattern are consistent with the idea that vacant myosin-binding sites on thin filaments are filled by exogenous S1. Electron microscopy indicates that S1 permeates and labels fibers and fibrils completely. Electron micrographs also show that cross-bridges are not displaced by exogenous S1 under the conditions used, and this is supported by the unchanged mechanical stiffness of the S1-labeled fibers. The amount of bound S1, as measured by gel electrophoresis and interference microscopy, together with the magnitude of the intensity changes in the x-ray diffraction pattern, is consistent with a thick filament structure that contains four molecules of endogenous myosin per 14.5 nm of its length, but does not agree well with earlier estimates of six myosins per crown. Lack of information on possible inhibition of S1-binding by factors other than the presence of cross-bridges, e.g., troponin, render uncertain calculations of the number of attached cross-bridges in the rigor state. However, it appears that at least 75% of the endogenous myosin heads are attached. Occupancy of binding sites on thin filaments after incubation with S1 is high, probably greater than 85%, so that x-ray scattering from those parts of the structure that adhere to the symmetry of the thin filaments can be treated as diffraction from S1-decorated thin filaments. In addition, we show in thin flared X cross sections that exo-S1 heads bind to actin with the geometry described in decorated actin by Taylor, K.A., and L.A. Amos.
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D004586:Electrophoresis; D066298:In Vitro Techniques; D007313:Insecta / Q000378:metabolism*; D008854:Microscopy, Electron; D009119:Muscle Contraction; D009132:Muscles / Q000378:metabolism* / Q000648:ultrastructure; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D014961:X-Ray Diffraction
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D009218:Myosins