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(Circulation Research. 1997;81:688-693.)
© 1997 American Heart Association, Inc.

Articles

Interaction of the Heavy and Light Chains of Cardiac Myosin Subfragment-1 With F-Actin

O.A. Andreev, , J. Borejdo

From the Department of Biochemistry and Molecular Biology, University of North Texas, Fort Worth, Tex.

Correspondence to O.A. Andreev, Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107. E-mail jborejdo{at}hsc.unt.edu

Abstract 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+HC_S1, cdLC1+actin+HC_S1, and cdLC1+two actins+HC_S1, 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.

Key Words: F-actin • cardiac muscle • cross-linking • polarization • fluorescence

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