title: Localization of a light-chain binding site on smooth muscle myosin revealed by light-chain overlay of sodium dodecyl sulfate-polyacrylamide electrophoretic gels.
abstract: The interactions of smooth muscle myosin and its light chains have been examined by incubating sodium dodecyl sulfate-polyacrylamide gels of myosin with radioactively labeled regulatory or essential light chains. The technique involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fixation with methanol and acetic acid followed by an extensive series of washes. The gel is incubated overnight with labeled light chains in the presence of bovine serum albumin and then washed extensively to remove unbound protein. Following staining and destaining, the gel is autoradiographed to reveal which protein bands have bound light chain. The myosin heavy chain was able to rebind labeled regulatory or essential light chains despite the harsh procedure described above. By fragmenting the myosin heavy chain proteolytically, we were able to determine the binding site for both types of light chains to be within the 26,000-Da COOH-terminal segment of smooth muscle subfragment 1 (S-1) or the 20,000-Da COOH-terminal segment of skeletal muscle S-1. The extent of binding was 0.1-0.4 mol of light chain/mol of S-1 heavy chain. No binding was observed to portions of the myosin molecule which do not contain this segment such as myosin rod, light meromyosin, S-2, or the NH2-terminal 75,000-Da segment of S-1.
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D015879:Myosin Subfragments; D010446:Peptide Fragments; D014357:Trypsin; D010206:Papain; D009218:Myosins