Properties of the alkali light-chain-20-kilodalton fragment complex from skeletal myosin heads

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Abstract
We have developed a rapid and reproducible procedure widely applicable to the preparation of pure aqueous solutions of the complex between an alkali light chain and the COOH-terminal heavy-chain fragments of skeletal myosin chymotryptic subfragment 1 (S-1) split by various proteases. It was founded on the remarkable ethanol solubility of these complexes. A systematic study of the ethanol fractionation of the tryptic (27K-50K-20K)-S-1 (A2) showed the NH2-terminal 27K fragment to behave like a specific protein entity being quantitatively precipitated at a relatively low ethanol concentration. Only the 20K peptide .sbd. A2 complex remained in solution when the S-1 derivative was treated with exactly 4 volumes of ethanol in the presence of 6 M guanidinium chloride. At a lower ethanol concentration, a soluble mixture of 50K and 20K peptides together with the light chain was obtained. The isolated 20K fragment-A2 system containing a 1:1 molar ratio of each component was investigated by biochemical and 1H nuclear magnetic resonance (NMR) techniques to highlight its structure and the interaction of the 20K heavy-chain segment with F-actin and with the light chain. During the treatment of the complex with .alpha.-chymotrypsin, only the 20K peptide was fragmented in contrast to its stability within the whole S-1. The binding of F-actin to the complex led, however, to a strong inhibition of its chymotryptic degradation. 1-Ethyl-3-[3-(dimethylamino)propyl] carbodiimide cross-linking of F-actin to the complex produced covalent actin-20K peptide only, the amount of which was lower relative to that observed with the entire split S-1. The binding constant of actin to the complex determined by sedimentation at various strengths was 100-fold weaker than for native S-1 and was similar to that reported previously for the 20K peptide preparation without light chain. The 1H NMR experiments indicated that the interaction of the 20K peptide-A2 complex with F-actin led to changes in the tertiary structure of the A2 light chain consequent on actin binding to the 20K peptide. We conclude that an actin-induced conformational change could be transmitted through the 20K segment to the homologous 150-residue COOH-terminal region of the alkali light chains. This feature may have implications in the mechanism of energy transduction by the actomyosin complex.
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