Interaction of the heavy and light chains of cardiac myosin subfragment-1 with F-actin.
The interaction of the heavy chain (HC) and the light chain (cdLC1) of cardiac S1 (cdS1) with F-actin was studied by cross-linking, Western blotting, and fluorescence polarization methods. Incorporation of cdLC1 in cross-linked products was examined by Western blots using the primary antibody against 71-74 residues of cdLC1. Cross-linking with zero-length, water-soluble reagent yielded three products with apparent molecular masses of 150, 160, and 210 kD. Like in the case of cross-linking of skeletal S1 with actin, these complexes included only HC of S1 and actin. The composition of the products were as follows: 150 kD, one HC of S1 cross-linked through a primary site (on the C-terminal of the 20-kD fragment) to the N-terminus of actin; 160 kD, one HC of S1 cross-linked through a secondary site (on the 50 kD fragment) to the N-terminus of actin; and 210 kD, one HC of S1 cross-linked through primary and secondary sites to two actins. Four additional products with apparent molecular masses of 66, 120, 185, and 235 kD contained cdLC1 and were identified as cdLC1 + actin, cdLC1 + HCS1, cdLC1 + actin + HCS1, and cdLC1 + two actins + HCS1, respectively. The same products were observed when cross-linking was performed in cardiac myofibrils incubated with cdS1. The production of cross-linked complexes of the heavy and light chain with actin decreased with an increase in the molar ratio of cdS1:actin. To test whether the orientation of myosin heads depended on a degree of occupation of thin filaments, myofibrils were irrigated with varying concentrations of cdS1. Fluorescence polarization measurements of cdS1 bound to individual I-bands revealed that the orientation depended on the concentration.
Andreev
O A
OA
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth 76107, USA.
Borejdo
J
J
D000199:Actins / Q000378:metabolism*; D000818:Animals; D003432:Cross-Linking Reagents; D009206:Myocardium / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism*; D011485:Protein Binding; D013552:Swine
D000199:Actins; D003432:Cross-Linking Reagents; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D018995:Myosin Heavy Chains