Influence of the regulatory light chain of fast skeletal muscle myosin on its interaction with actin in the presence and absence of ATP.

The effect of myosin LC2 modifications (phosphorylation or selective proteolytic removal of a seven-residue N-terminal peptide) and partial or complete removal of the whole LC2 was studied under various conditions. (1) Actin binding in the absence of ATP is not influenced by the nature of the myosin species (phosphorylated, dephosphorylated or devoid of LC2). (2) A 50% inhibition of K+/EDTA-ATPase was obtained with actin concentrations hardly different when phosphorylated and dephosphorylated myosins were compared (of the order of 5 microM), whereas both myosin devoid of LC2 and myosin in which the LC2 N-terminal peptide has been removed required significantly higher concentrations of actin (13.0 +/- 2 and 12.0 +/- 2.0 microM, respectively). (3) Dissociation of the actomyosin complex at high ionic strength with nucleotides is not influenced by phosphorylation. (4) Actin activation of Mg2+-ATPase is enhanced when LC2 is phosphorylated; no activation enhancement is observed with myosin devoid of LC2. (5) Translational diffusion coefficient measurements of myosin in high-ionic-strength solutions indicate a tendency for LC2-deprived myosin to form autoassociation oligomers. It thus appears that a structural modification (partial cleavage or removal of LC2) induces important structural changes in myosin, pointing to a role for LC2 in the intrinsic conformation of the molecule and its interaction potentialities. Effects of LC2 removal at high ionic strength are best explained by interactions bearing no relationship to physiological functions. A physiologically significant effect of LC2 phosphorylation requires a minimum degree of organization (actomyosin complex) to be expressed in which LC2 could play the role of a return-spring in the cross-bridge mechanism.

The influence of ethylenediaminetetraacetate on white skeletal muscle myosin.

Myosin from rabbit white skeletal muscle was treated with 10 mM EDTA in 150 mM phosphate buffer. After precipitation of myosin by dialysis against a 14-fold volume of water, EDTA-treated myosin, myosin before treatment and the supernatant from the treatment of myosin with EDTA were examined on sodium dodecyl sulphate-polyacrylamide gels by electrophoresis. It has been found that the quantity of LC2 light chains diminished after treatment with EDTA, and the supernatant contained the LC2 light chains. Treatment of myosin with EDTA in the presence of Mg2+ does not change the stoichiometry of the LC2 light chain and the supernatant is free from LC2 light chains. The treatment of myosin with p-chloromercuri-benzoate leads to dissociation of the same amount of LC2 light chains. It is suggested that divalent cations and thiol groups are engaged in the attachment of LC2 light chain to the myosin molecule.

The binding of actin to phosphorylated and dephosphorylated myosin.

The binding of actin to myosin containing phosphorylated and dephosphorylated light chains (LC2) was investigated by studying the influence of actin on Mg2+- and K+-stimulated ATPase of phosphorylated and dephosphorylated myosin and by comparing the influence of PPi on actomyosin formed from pure actin and phosphorylated or dephosphorylated myosin. The concentration of actin producing inhibition of one half of myosin K+-ATPase activity was 4.1 micro M and 7.7 micro M for phosphorylated and dephosphorylated myosin, respectively. Actomyosin formed from dephosphorylated myosin dissociated at lower PPi concentration than did that from the phosphorylated form. The extrapolated values of Km obtained from studies of the influence of actin on Mg2+-ATPase activity of dephosphorylated myosin were about twice as high as for the phosphorylated form. Thus, the affinity of phosphorylated myosin for actin was significantly higher under conditions studied.

The phosphorylation-dephosphorylation process as a myosin-linked regulation of superprecipitation of fast skeletal muscle actomyosin.

The dependence of the onset and course of turbidity changes ( superprecipitation) induced by ATP were studied in a natural actomyosin suspension with the dephosphorylated and phosphorylated forms of light chains (LC2) of myosin. It was found that the onset and time course of the changes in turbidity of the natural actomyosin suspension are strongly dependent on the (phosphorylated and dephosphorylated) form of these chains of myosin. The ATPase activity of actomyosin with phosphorylated LC2 was lower and the half-time for achieving maximal turbidity of actomyosin suspension after addition of ATP was higher than that of actomyosin with dephosphorylated LC2. Natural actomyosin preparations contain endogenous light-chain kinase and phosphatase. The changes of turbidity induced by ATP in the natural actomyosin suspension are greatly diminished in the presence of phosphate. Thiophosphorylation of LC2 of myosin leads to a decrease of the extent of superprecipitation of natural actomyosin. The release of [32P]phosphate from actomyosin containing [32P]ATP-phosphorylated LC2 of myosin increases with increased turbidity of actomyosin suspension. The change of the form LC2 as a kind of additional myosin-linked regulation of superprecipitation is discussed.

Factors influencing interaction of phosphorylated and dephosphorylated myosin with actin.

The influence of various factors on the interaction of phosphorylated and dephosphorylated myosin with actin was examined. It was found that the difference between the values of specific activity of the two myosin forms of actin-stimulated Mg2+-ATPase is affected by changes in KCl, MgATP and actin concentration. The effect of increased pH on the differences in the rate of ATP hydrolysis by actomyosin containing phosphorylated myosin as compared with that of the dephosphorylated one, observed in the presence of EGTA, is abolished by addition of Ca2+. Tropomyosin strongly inhibits the actin-stimulated Mg2+-ATPase of phosphorylated myosin (by about 60%). The tropomyosin-troponin complex and native tropomyosin lowered the rate of ATP hydrolysis by actomyosin containing both phosphorylated and dephosphorylated myosin by about of 60% of the value obtained in the absence of those proteins. These results indicate that the change of negative charge on the myosin head due to phosphorylation and dephosphorylation of myosin light chains modulates the actin-myosin interaction at different steps of the ATP hydrolysis cycle. Phosphorylation of myosin seems to be a factor decreasing the rate of ATP hydrolysis by actomyosin under physiological conditions.

The possible role of myosin A1 light chain in the weakening of actin-myosin interaction.

The effects resulting from the removal of the N-terminus of myosin A1 by limited papain cleavage are investigated. The myosin and heavy meromyosin K+-ATPase and Ca2+-ATPase activities, and actin-activated ATPase activity of heavy meromyosin (HMM) and subfragment-1, are studied. Myosin and HMM preparations devoid of the A1 N-terminus exhibits lower Ca2+-ATPase activities at low ionic strength whereas no differences in K+- or Ca2+-ATPase activities are observed at high ionic strength. Direct binding of actin to monomeric myosin under K+-activated ATPase conditions is much more effective for myosin containing a shortened A1 light chain. The kinetic constants K(app) for actin and V(max) are calculated from actin-activation curves for HMM and subfragment-1. The kinetic constants for HMM are determined under conditions assuring saturation of regulatory light chains (RLC) either with Mg2+ or Ca2+. The removal of the A1 N-terminus influences the actin-myosin interaction in a Ca2+- and phosphorylation-dependent manner; in most cases, this leads to an increase in affinity. In the case of subfragment-1, the removal of the N-terminus of A1 led to a decrease in affinity. It is reasonable to assume that the intact A1 light chain may cause weakening of the actin-myosin interaction under certain conditions. This weakening may be regulated by RLC phosphorylation and RLC-bound calcium-for-magnesium exchange. Such an effect requires a structural minimum that is present in HMM but not in subfragment-1. Implications of such a role for the A1 N-terminus in the myosin-actin interaction are discussed.

Decoration of actin filaments with skeletal muscle heavy meromyosin containing either phosphorylated or dephosphorylated regulatory light chains.

Heavy meromyosin containing almost intact regulatory light chains (LC2) was obtained from monomeric phosphorylated and dephosphorylated rabbit fast skeletal muscle myosin by brief chymotryptic digestion in the presence of CaCl2. Actin filaments, complexed with heavy meromyosin, display two different forms of arrowhead, depending on the form of LC2.

Skeletal muscle myosin regulatory light chains conformation affects the papain cleavage of A1 light chains.

In the present study, the influence of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (RLC) on accessibility of myosin and heavy meromyosin alkali light chains (A1) for papain digestion was investigated. The properties of native and papain treated myosin and heavy meromyosin were compared. Exchange of magnesium ions bound to RLCs for calcium ions accelerates the digestion of A1 in the presence of ATP in dephosphorylated myosin, heavy meromyosin, acto-myosin and the acto-heavy meromyosin complex. In the absence of ATP the exchange of magnesium ions bound to RLCs for calcium ions delays the digestion of A1 in the acto-myosin complex. Myosin and heavy meromyosin having shortened A1 by papain cleavage shows decreased K(+)-ATPase and increased actin binding ability in the presence and absence of ATP. The cooperation of RLC and A1 with heavy chains in the changes of structural organization of myosin head during muscle contraction is discussed.

Dual effect of actin on the accessibility of myosin essential light chain A1 to papain cleavage.

The influence of various amounts of actin on the proteolytic susceptibility of myosin essential light chain (ELC) A1, the binding of isolated A1 light chain and the N-peptide spanning N-terminal sequence of A1 to actin is studied to obtain more information on the role of the N-terminus of A1 light chain in the myosin-actin interaction. Low ratios of actin to myosin (1:1) lead to stimulate cleavage, whereas higher ratios (4:1) lead to protection of A1. Exposure of A1 by actin is especially seen in heavy meromyosin (HMM) and monomeric myosin and this is probably related to the full saturation of actin protomers with myosin heads. The protecting action of actin on A1 cleavage is more pronounced in myosin filaments. Conditions favoring the saturation of myosin regulatory light chain (RLC) with calcium ions instead of magnesium ions promotes the protection of A1. Cross-linking of HMM and actin results in higher yields of A1-actin product at high actin to myosin heads ratios. Isolated A1 light chain is pelleted by actin. A synthetic peptide spanning the N-terminal sequence of A1 can be cross-linked to actin. It is postulated that the protective action of actin on A1 papain cleavage is caused by the binding of the A1 N-terminus to actin. Changes in the RLC phosphorylation level and magnesium-for-calcium exchange in RLC may affect the probability of this interaction.

Conformational changes of F-actin in myosin-free ghost single fibre induced by either phosphorylated or dephosphorylated heavy meromyosin.

The changes in F-actin conformation of myosin-free single ghost fibre induced by binding of phosphorylated or dephosphorylated heavy meromyosin have been studied by measuring polarized fluorescence of F-actin intrinsic tryptophan and of phalloidin-rhodamine bound to F-actin. The changes of polarization of both fluorescences were found to be dependent on low or high Ca2+ concentration and on the phosphorylated or dephosphorylated form of heavy meromyosin. Computer analysis of polarized fluorescence has shown that binding of phosphorylated heavy meromyosin with divalent ion binding sites saturated with Mg2 (in the presence of 1 mM MgCl2 and 1 mM EGTA) and dephosphorylated heavy meromyosin with divalent ion binding sites saturated with Ca2+ (in the presence of 1 mM MgCl2 and 0.1 mM Ca2+) decreases the angles of emission and absorption dipoles and the angle between the F-actin axis and the fibre axis, thus suggesting that F-actin in ghost fibre becomes more flexible. On the other hand, the above-mentioned angles increase when phosphorylated heavy meromyosin at high and dephosphorylated heavy meromyosin at low Ca2+ concentration were bound to thin filaments, thus showing the decrease of F-actin flexibility under these conditions.

Binding of phosphorylated and dephosphorylated heavy meromyosin to F-actin.

The effect of myosin light chain phosphorylation in skeletal muscle was investigated with respect to the binding affinity of phosphorylated and dephosphorylated heavy meromyosin (HMM) for F-actin in the absence of ATP. For phosphorylated HMM the affinity was 2.5-times weaker in the presence of Ca2+ as in its absence (HMM divalent binding sites saturated only with Mg). For dephosphorylated HMM the reverse was true, the binding being 2.4-times higher in the presence of Ca2+.

Influence of Mg2+ and Ca2+ bound to 1,5-IAEDANS-labeled phosphorylated and dephosphorylated heavy meromyosin complexed with F-actin on polarized fluorescence of the fluorophore.

Dephosphorylated and phosphorylated heavy meromyosin, fluorescently labeled with 1,5-IAEDANS attached at the SH1 group, was introduced into myosin-free ghost fibres and the polarized fluorescence of the bound label was measured. The results depended on whether the divalent cation binding sites on heavy meromyosin were saturated with Mg2+ or Ca2+. The calculated angles of absorption and emission dipoles and the amount of random fluorophores were significantly changed, indicating that the random mobility and orientation of the fluorophores of phosphorylated and dephosphorylated heavy meromyosin heads complexed with F-actin in the ghost fibre depend on saturation of heavy meromyosin with Ca2+ or Mg2+. The presence of bound Ca2+ has an opposite effect on the polarized fluorescence of phosphorylated and dephosphorylated 1,5-IAEDANS-heavy meromyosin.

Regulatory light chain influences alterations of myosin head induced by actin.

The effect of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (LC2) on structural organization of rabbit skeletal myosin head was studied by limited tryptic digestion. In the presence of actin, exchange of magnesium bound to LC2 by calcium in dephosphorylated myosin accelerates the digestion of myosin and heavy meromyosin heavy chain and increases the accumulation of a 50 kDa fragment. This effect is significantly diminished in the case of phosphorylated myosin. Thus, both phosphorylation and cation exchange influences the effect of actin binding on the structural organization of myosin head.

Conformational changes of contractile proteins accompanying modulation of skeletal muscle contraction. Polarized microfluorometry investigations.

Results of studies on the modulation of skeletal muscle contraction by phosphorylation of myosin regulatory light chains and by exchange of magnesium for calcium in myosin heads were reviewed. The polarized fluorescence method was used in these studies, and conformational changes of contractile proteins accompanying modulation of skeletal muscle contraction were investigated. It was found that both the exchange of bound magnesium for calcium on myosin heads and the phosphorylation of myosin regulatory light chains control the ability of myosin heads to induce, upon binding to actin, conformational changes of thin filament leading to decrease or increase of its flexibility. The changes in actin filament flexibility may be caused by alteration of both the inter- and the intramonomer structural organization.

Some properties of glycerinated skeletal muscle fibers containing phosphorylated myosin.

The structural changes of phalloidin-rhodamin labelled F-actin at relaxed and contracted skeletal muscle fibre containing phosphorylated myosin and at contracted state after dephosphorylation were investigated by measuring of polarized fluorescence of the fluorophore. The mechanical properties (isometric tension development) of fibre were studied in parallel. At submaximal concentration of Ca ions (0.6 mumol/l) the isometric tension was decreased after dephosphorylation of fibre myosin. The changes in polarization of fluorophore bound to actin filament were correlated with isometric tension developed by the muscle fibre. The angles between the actin filament long axis and the absorption and emission dipoles for contracted and relaxed fibre were different, suggesting changes in the organization of the actin monomers in thin filament, dependent on the physiological state of the fibre. The flexibility of the thin filaments during transition of the fibre from relaxed to "contracted" state increases as indicated by greater average angle between the F-actin long axis and the fibre axis.

[The disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent]. / Ischeznovenie zavisimosti aktin-miozinovogo vzaimodeistviia ot fosforilirovaniia legkikh tsepei miozina pri "zamorazhivaniia" struktury tiazhelogo meromiozina bifunktsional'nym reagentom.

Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.