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 cdLC 1 in cross-linked products was examined by Western blots using the pri mary antibody against 71-74 residues of cdLC1. Cross-linking with zero -length, water-soluble reagent yielded three products with apparent mo lecular masses of 150, 160, and 210 kD. Like in the case of cross-link ing of skeletal S1 with actin, these complexes included only HC of S1 and actin. The composition of the products were as follows: 150 kD, on e HC of S1 cross-linked through a primary site (on the C-terminal of t he 20-kD fragment) to the N-terminus of actin; 160 kD, one HC of S1 cr oss-linked through a secondary site (on the 50 kD fragment) to the N-t erminus of actin; and 210 kD, one HC of S1 cross-linked through primar y and secondary sites to two actins. Four additional products with app arent molecular masses of 66, 120, 185, and 235 kD contained cdLC1 and were identified as cdLC1+actin, cdLC1+HCS1, cdLC1+actin+HCS1, and cdL C1+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 chai n with actin decreased with an increase in the molar ratio of cdS1:act in. To test whether the orientation of myosin heads depended on a degr ee of occupation of thin filaments, myofibrils were irrigated with var ying concentrations of cdS1. Fluorescence polarization measurements of cdS1 bound to individual I-bands revealed that the orientation depend ed on the concentration.