Three-dimensional reconstruction of thin filaments decorated with a Ca2+-regulated myosin
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Tropomyosin
Helix (gastropod)
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Heavy chain
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The myosin regulatory and essential light chains in skeletal muscle do not play a role as significant as in scallop or smooth muscle, however, there are some data suggesting that the skeletal myosin light chains and their N‐terminal parts may have a modulatory function in the interaction of actin with myosin heads. In this paper four conformational states of the myosin head with respect to the regulatory light chain bound cation (magnesium or calcium) and phosphorylation are proposed. Communication between regulatory and essential light chains and putative binding of the N‐terminus of A1 essential light chain to actin is discussed.
Meromyosin
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It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the β-chain or Cys190 of the α-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.
Tropomyosin
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An antibody with specificity for the 20 kDa myosin light chain of smooth muscle phosphorylated by myosin light chain kinase was developed. The antibody was raised against the phosphorylated synthetic peptide, Lys-Lys-Arg-Pro-Gln-Arg-Ala-Thr-phospho-Ser-Asn-Val-Phe-Cys (residues 11-22 of the myosin light chain). This antibody only recognized the monophosphorylated myosin light chain at serine 19, i.e., with no detectable recognition of nonphosphorylated or diphosphorylated serine 19 or threonine 18. The specificity was tested by EIA assaying of myosin light chain kinase activity using a 96-well plate coated with the light chain. This EIA system was as sensitive as the radioisotopic method, and the newly developed non-radioisotopic method.
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Abstract The phosphorylatable light chain of myosin (P‐light chain) from hearts of the turtle, rat, frog, dog, cat, adult chicken and 1 week old chick was compared by two dimensional gel electrophoresis. The technique separates the phospho and dephospho forms of P‐light chain and allows quantitation of its percentage distribution. The P‐light chain was found to exist in multiple forms. The turtle heart possessed the highest percentage, 75%, of the phospho form of P‐light chain, whereas 25% of P‐light chain was in the dephospho form. In rat heart, 35% of the P‐light chain was phosphorylated and 65% dephosphorylated. In frog heart, the P‐light chain exhibited three forms: two phosphorylated and one dephosphorylated. The P‐light chain of dog, cat and adult chicken hearts displayed four spots on two‐dimensional gel electrophoresis: two phospho and two dephospho forms. In these species the percentage of total P‐light chain being in the phospho forms was in the range of 10–25%. Comparison of adult chicken and 1 week old chick preparations suggests that during maturation from the chick to the chicken the percentage phospho form of the total P‐light chain decreases. The myosin light chain kinase and phosphatase activities in whole homogenates of the above heart preparations were assayed. The data, with the exception of those from frog heart, suggest that the extent of light chain phosphorylation is correlated with the ratio of light chain kinase to phosphatase activity.
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The segment of smooth muscle regulatory light chain essential for the phosphorylation dependent activation of actomyosin motor activity and the binding of myosin heavy chain was identified. The C-terminal domain of the 20-kDa light chain, which is less conserved than the rest of the polypeptide among various muscle types, was mutated by either deletion or substitution of amino acid residues and the mutant light chains were then incorporated into myosin by subunit exchange. Deletion of Lys149-Ala166 markedly reduced the affinity of the light chain for the heavy chain, whereas the C-terminal five residues, Lys167-Asp171, did not contribute to the binding affinity. Deletion of Lys149-Phe158 abolished the phosphorylation-dependent activation of actomyosin ATPase activity as well as superprecipitation activity. These results suggest that the C-terminal domain of the regulatory light chain is critical for transmitting the change in the conformation of the regulatory light chain induced by phosphorylation at Ser19 to the heavy chain.
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Meromyosin
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Meromyosin
Heavy meromyosin
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