Interaction of the heavy and light chains of cardiac myosin subfragment- 1 with F-actin

O. A. Andreev, J. Borejdo

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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 t- bands revealed that the orientation depended on the concentration.

Original languageEnglish
Pages (from-to)688-693
Number of pages6
JournalCirculation research
Volume81
Issue number5
DOIs
StatePublished - 1997

Keywords

  • Cardiac muscle
  • Cross-linking
  • F-actin
  • Fluorescence
  • Polarization

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