Metal binding to myosin and to myosin DTNB-light chain

Mm, Werber

doi:10.1007/bf01936967

Cited by 13 publications

(6 citation statements)

References 20 publications

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“…In intact myosin or its fragments these changes are largely masked by the background due to the heavy chains. A fluorescence change on calcium or magnesium binding can indeed be detected, as previously shown by Werber [18], but the effect is too small to permit its use for the construction of accurate titration curves, at least with a single-beam fluorimeter. The change in a-helix content, seen in circular dichroism, is also obscured by the vastly larger ellipticity arising from the heavy chain.…”

Section: Resultsmentioning

confidence: 71%

“…If binding is accompanied by a spectroscopic change, this is more likely to fulfil the requirements in this regard. It is known that the binding of calcium and magnesium ions to isolated skeletal myosin metal-ion binding light chains, leads to a quenchingof the intrinsic fluorescence of the protein [4,17,18], an increase in the negative ellip-ticity in the region of the peptide Cotton effects and an appreciable change in the system of small Cotton effects associated with aromatic side chains [4], as well as hydrodynamic changes [4]. In intact myosin or its fragments these changes are largely masked by the background due to the heavy chains.…”

Section: Resultsmentioning

confidence: 99%

“…The precision is sufficient to yield linear plots (in effect Scatchard plots) if the ratio of fractional saturation to free metal ion concentration is plotted against fractional saturation. The shapes of the binding curves suggest that we can also disregard any contribution from weak binding sites (which are undoubtedly present on the light chains [18,19]). …”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation and the Binding of Calcium and Magnesium to Skeletal Myosin

Kardami¹,

Alexis²,

Paz³

et al. 1980

European Journal of Biochemistry

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It has previously been shown that the binding of calcium and magnesium ions to the isolated metalbinding light chains, i.e. those dissociable by 5,5'-dithiobis(2-nitrobenzoate), of rabbit skeletal muscle myosin is moderated by phosphorylation and is accompanied by a sizeable conformational change. As judged by circular dichroism in the region of the aromatic Cotton effects, this conformational change occurs when calcium ions bind to the light chain in situ on the myosin head. Moreover the affinity for calcium is again changed by phosphorylation. The change in chymotryptic digestion patterns, in particular the protection of the head-rod junction in insoluble myosin, by divalent cations, has been used to obtain binding profiles. The results are consistent with the presence of a single class of independent sites, showing no cooperativity. The affinity of the site for both calcium and magnesium ions is enhanced by 1-2 orders of magnitude when the light chain is incorporated in the myosin heads. The effect of phosphorylation on the affinity persists in these circumstances, being marked for calcium and small for magnesium. On phosphorylation the calcium binding constant falls from 8 x lo6 M-' to 4 x lo6 M-' at physiological ionic strength, compared with 2.5 x lo5 M-' and 5 x lo4 M -for the isolated light chains. The sensitivity of the proteolytic cleavage sites is affected by phosphorylation. Thus in the absence of calcium ions the yield of subfragment 1 at a low chymotrypsin concentration is substantially greater in dephosphorylated than phosphorylated myosin, whereas at saturating concentrations of calcium ions attack at the light meromyosin/heavy meromyosin junction is favoured by phosphorylation. These observations may signify a structural effect of phosphorylation on the prevailing interactions within the myosin filament in physiological solvent conditions. It has become clear that the calcium ions, which are released into the sarcoplasm from the sarcoplasmic reticulum when a muscle is stimulated, have multifarious effects. In vertebrate striated muscle, calcium ions are captured by troponin C, with relief of the inhibition (exerted by the regulatory complex on the thin filaments) of the actomyosin interaction. In molluscan muscle, a homologous protein is present as a subunit (regulatory light chain) on the myosin heads, and this is the site of the primary control mechanism; in some other muscles both forms of regulation coexist (see Lehman and Szent-Gyorgyi [l] and Perry [2] for reviews). In smooth muscle, moreover, the ATPase activity is generally held to be regulated by phosphorylation of the calcium-binding myosin light Abbreviations. Nbsz light chains, the metal-binding light chains which can be dissociated from myosin by treatment with 53'-dithiobis(2-nitrobenzoate); Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid; EPR, electron paramagnetic resonances. chain, through the action of a kinase, which is itself activated by calcium ions. A similar phosphorylation reaction operates in striated muscle...

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Section: Resultsmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Phosphorylation and the Binding of Calcium and Magnesium to Skeletal Myosin

Kardami¹,

Alexis²,

Paz³

et al. 1980

European Journal of Biochemistry

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“…Ca 2ϩ -induced conformational changes. In heavy meromyosin from vertebrate skeletal muscle, Ca 2ϩ causes a 6% decrease in tryptophan fluorescence (27), as well as a small blue shift (28). Since neither of these effects occurred with the isolated heavy chains (29) and the quenching was accentuated with the isolated light chains, the authors concluded that the fluorescence changes in heavy meromyosin were caused by conformational changes in the light chains only.…”

Section: Discussionmentioning

confidence: 99%

Calcium-Induced Quenching of Intrinsic Fluorescence in Brain Myosin V Is Linked to Dissociation of Calmodulin Light Chains

Cameron

Carvalho

Araújo

et al. 1998

Archives of Biochemistry and Biophysics

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“…Light chain LC2 is more firmly attached to myosin in the presence of Ca2+ (Szent-Gyorgyi, 1975;Kasman & Kadol, 1977). When in association with the heavy chains the binding of Ca2+ is weaker at pH 6.5 as compared with pH 7.4 (Wikman-Coffelt, Muhlrad et al, 1962;Werber, 1978). H+ may induce conformational changes in the swivel region of the heavy chains (Mendelson & Cheung, 1976;Chiano & Harrington, 1979), whereas pH has no influence on the conformation of the dissociated light chains (Werber, 1978; Stafford & Szent-Gyorgyi, 1978).…”

mentioning

confidence: 99%

Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2

Srivastava

Mühlrád

Wikman‐Coffelt

1981

Biochemical Journal

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The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca(2+)-binding properties of the light chains. The Ca(2+)-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH(2) thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca(2+)-binding properties of light chains and generation of tension. When the SH(2) moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca(2+)-binding properties of light chain LC(2) is lost; under these conditions the Ca(2+)-binding affinity value of SH(2)-N-ethylmaleimide-blocked myosin (3.3x10(4)m(-1)) decreases to near that expressed with the dissociated light chain LC(2) (0.7x10(4)m(-1)). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH(2) thiol does not affect Ca(2+) binding. The native secondary and tertiary structure of myosin seem to be required for Ca(2+) binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH(2)-N-ethylmaleimide-blocked myosin normal Ca(2+)- and (Mg(2+)+actin)-stimulated ATPase activities are expressed; however, there is a loss in K(+)-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca(2+) with alterations in pH values. In the absence of Ca(2+)/EGTA buffer the biphasic Ca(2+)-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2x10(6)m(-1) and site two: 0.4x10(6)m(-1)) as compared with values obtained at pH6.5 (site one: 0.64x10(6)m(-1) and site two: 0.2x10(6)m(-1)). The Ca(2+)-binding affinity of light chain LC(2) and S(1), where the (S-1)-(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca(2+)-binding affinity, approx. 0.7x10(4)m(-1), whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca(2+)-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S(1) myosin subfragment light chain LC(2) was lost and thus was added back to the purified S(1) fraction. Light chain LC(2) was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)-(S-2) junction is needed for the positioning of light chain LC(2) and thus influences its essential conformation for Ca(2+) binding.

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Metal binding to myosin and to myosin DTNB-light chain

Cited by 13 publications

References 20 publications

Phosphorylation and the Binding of Calcium and Magnesium to Skeletal Myosin

Phosphorylation and the Binding of Calcium and Magnesium to Skeletal Myosin

Calcium-Induced Quenching of Intrinsic Fluorescence in Brain Myosin V Is Linked to Dissociation of Calmodulin Light Chains

Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2

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