Analysis of the metal-induced conformational change in myosin with a monoclonal antibody to light chain two.
A monoclonal antibody capable of detecting a conformational change in myosin light chain two (LC2) was characterized in detail. The antibody was shown to bind only to myosin LC2 when tested against fast skeletal myosin (chicken pectoralis muscle). With cardiac or slow muscle myosins, the antibody exclusively recognized their first light chains (LC1). Staining of myofibrils by the monoclonal antibody could be observed only after their irreversible denaturation by acetone or ethanol, or after incubation of the myofibrils in divalent metal chelators. This latter effect was shown to be fully reversible. The metal effect was independent of ionic strength although the affinity of the antibody for myosin was depressed at high salt concentrations. Similar metal effects were detected in the binding of antibody to cardiac or slow myosins. Neither the metal nor the ionic strength-related inhibition of antibody binding were detected with denatured myosin. The antibody binding site overlaps one of the alpha-chymotryptic sites in LC2 protected by divalent metals. Electron microscopic observations of myosin-antibody complexes demonstrated that the antibody binding site is located near the head-rod junction of myosin. Since the binding site of this monoclonal antibody has been mapped by recombinant DNA methods to the junction of the first alpha-helical domain with the calcium binding site of LC2, the location of the calcium binding site must also be located near the head-tail junction of myosin. A model for conformational changes at the myosin head-tail junction is proposed to account for the metal-induced blockage of antibody binding and the inhibition of alpha-chymotryptic digestion of LC2.
D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D007525:Isoelectric Focusing; D008274:Magnesium; D008854:Microscopy, Electron; D009210:Myofibrils / Q000032:analysis; D009218:Myosins / Q000276:immunology; D009994:Osmolar Concentration; D011487:Protein Conformation; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D009218:Myosins; D008274:Magnesium; D002118:Calcium