[Modulation of S-1 conformation and inhibition of the skeletal muscle S-1-ATPase by calponin of the mussel].

A novel 40 kDa protein has been detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. In this study, using skeletal muscle actin and S-1, we investigated the effects of the mussel 40-kDa actin-binding protein on the acto · S-1 ATPase activity. On increasing the 40-kDa actin-binding protein (CaP-40) concentration, the actin-activated ATPase activity decreased, and was inhibited 80% at a CaP-40 to actin ratio of 0.

[Abnormal tropomyosin function in ATPase cycle in hypertrophic and dilated cardiomyopathies].

Pathogenesis of most myopathies including inherited hypertrophic (HCM) and dilated (DCM) cardiomyopathies is based on modification of structural state of contractile proteins induced by point mutations, such as mutations in alpha-tropomyosin (TM). To understand the mechanism of abnormal function of contractile system of muscle fiber due to Glu180Gly, Asp175 or Glu40Lys, Glu54Lys mutations in alpha-TM associated with HCM or DCM, we specifically labeled alpha-TM by fluorescence probe 5-IAF after Cys-190 and examined the position and mobility of the IAF-TM in the ATP hydrolysis cycle using polarized fluorescence technique. Analysis of the data suggested that the point mutations in alpha-TM associated with hypertrophic or dilated cardiomyopathy caused abnormal changes in the affinity ofTM to actin and in the position of this protein on the thin filaments in the ATPase cycle.

[Hyperthyreosis inhibits the ability of actin to form strong-binding with myosin].

The effect of hyperthyreosis development induced by the increase in thyroid hormones in rats (during 2-4 weeks) on the orientation and mobility of fluorescent probe N-(iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine) specifically bound to Cys 374 of actin in ghost muscle fibers isolated from fast (EDL) and slow (SOL) rat muscles was studied. It was found that the binding of myosin subfragment-1 (S1) to F-actin induced the typical for the formation of strong binding actomyosin decrease in mobility of actin subdomain 1 and its rotation towards thin filament periphery. Development of hyperthyreosis markedly inhibited these phenomena.

[Effect of hypothyreosis on actin subdomain-1 movement induced by myosin subfragment 1-binding in fast and slow rat skeletal muscles].

Orientation and mobility of fluorescent probe N-((iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine)(1.5-IAEDANS)) specifically bound to Cys-374 of actin in ghost muscle fibers isolated from fast and slow rat muscles were studied by polarized fluorimetry in the absence and presence of myosin subfragment-1 (S1) in intact rats and in the animals with gradual (during 2-5 weeks) reduction of thyroid hormones synthesis (hypothyreosis development). S1 binding to F-actin of ghost muscle fibers was shown to induce changes in orientation of the dipoles of the fluorescent probe 1.

[The influence of caldesmon on strong binding of myosin with actin in denervated rat skeletal muscles].

The effect of caldesmon (CaD) on conformational changes in F-actin modified by fluorescent probe TRITC-phalloidin was investigated by polarized fluorimetry. Changes were induced by a subfragment-1 (S-1) of myosin in the absence or presence of CaD in ghost muscle fibers obtained from intact and denervated slow (SOL) and fast (EDL) skeletal muscles of rats. S-1 binding to actin of both SOL and EDL muscles was shown to cause changes in polarized parameters of TRITC-phalloidin typical for a strong actin-myosin binding as well as of transition ofactin subunits from "off" to "on" state.

[C-terminal sites of caldesmon drive ATP hydrolysis cycle by shifting actomyosin itermediates from strong to weak binding of myosin and actin].

Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle.

[Conformational changes of actin induced by strong or weak myosin subfragment-1 binding].

Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid.

[Effect of the C-terminal actin-binding sites of caldesmon on the interaction of actin with myosin].

TRITC-phalloidin or FITC-labeled F-actin of ghost muscle fibers was bound to tropomyosin and C-terminal recombinant fragments of caldesmon CaDH1 (residues 506-793) or CaDH2 (residues 683-767). After that the fibers were decorated with myosin subfragment 1. In the absence of caldesmon fragments, subfragment 1 interaction with F-actin caused changes in parameters of polarized fluorescence, that were typical of "strong" binding of myosin heads to F-actin and of the "switched on" conformational state of actin.

[Cleavage of DNA-binding loops of actin by subtilisin prevent formation of a strong type of myosin binding with actin].

In order to elucidate the role of DNA-binding loop of actin (amino acid residues 38-52) in mechanisms of muscle contraction, polarizational fluorimetry and ghost muscle fibers, containing thin filaments reconstructed by intact and subtilisin-cleaved G-actin were used. The thin filaments were modified by fluorescent probes rhodamin-phalloidin and 1,5-IAEDANS. Changes in orientation and mobility of the probes were considered as an indication of changes in actin conformation.

[Caldesmon inhibits formation of strongly bound myosin cross-bridges and activates an ability of weakly bound cross-bridges to transform actin monomers to the off-conformation].

The effect of caldesmon and its actin-binding C-terminal 35 kDa fragment on conformational alterations of actin in a muscle fiber at relaxation, rigor and at simulation of strong and weak binding of myosin heads to actin was studied by polarizational fluorimetry technique. The strong and weak binding forms were mimicked during binding of F-actin of ghost muscle fibers to myosin subfragment-1 modified with NEM (NEM-S1) or pPDM (pPDM-S1), respectively. As a test for alterations in actin conformation, changes in orientation and mobility of a fluorescent probe, TRITC-phalloidin, bound specifically to F-actin were used.

[Study of molecular mechanisms of muscle contraction using polarization fluorometry].

The review summarizes results of studies on the conformational changes in contractile proteins during muscle contraction. The studies were carried out by polarized fluorescence technique in the UV and visible light. The revealed were alterations of actin and myosin in muscle fiber, taking place at various stages of contractile cycle.

[The interaction of actin with myosin in fast and slow muscles of the mouse].

Conformational changes of actin, during the transition of glycerinated muscle fibers of fast (EDL) and slow (SOL) mouse muscles from relaxation to rigor, were investigated by the polarized fluorescent technique. Changes in orientation and mobility of the fluorescent probe, i.e.

[The effect of calponin on the rate of actin filament movement].

The effect of calponin on the velocity of actin filaments sliding over skeletal and phosphorylated smooth myosins was studied by in vitro mobility assay. It was found that calponin, being part of an actin filament, inhibits the average velocity of thin filaments movement. The analysis of histograms of the velocities showed that in the presence of calponin, actin filaments are capable to slow down the sliding, stop moving and move with high velocity, characteristic of calponin-free filaments.

[The effect of a caldesmon fragment with a mol. weight of 38 kDa and calponin on the capacity of actin to form a "strong" form of binding with myosin heads].

Effect of calponin and 38 kD actin-binding proteolytic fragment of caldesmon on actin structure alterations, initiated by decoration of thin filaments by N-ethylmaleimide-modified skeletal myosin subfragment-1 (NEM-S1) and by phosphorylated smooth heavy meromyosin (pHMM), has been studied by polarized fluorimetry. F-actin of myosin-free ghost fiber was labeled with fluorescent probe fluoroscein-5-maleimide. Both the actin-binding regulatory proteins have been demonstrated to inhibit conformational changes of actin typical for the "strong" binding of myosin head to actin.

[Effect of phosphorylating myosin light chains and ionic strength on actin-myosin interaction in a relaxed skeletal muscle fiber].

The effect of LC-2 phosphorylation and ionic strength on actin-myosin interaction in relaxed skeletal muscle fibers have been studied using polarization fluorimetry. F-actin was chemically modified by the fluorescent dye, rhodamine-phalloidin, and the mode of myosin-actin interaction was estimated by a polarized fluorescence technique based on changes in the dye orientation (phi E) and thin filament flexibility (sin 2 theta). Phosphorylation of LC-2 at relaxation in low ionic strength induced typical for the force production ("strong" binding) state changes in the polarized fluorescence of F-actin (decreasing of phi E and increasing of sin 2 theta).

[Calponin inhibits the strong type of myosin binding with actin].

The effects of calponin on conformational changes in actin caused by modelling of "strong" binding between actin and myosin heads have been studied using polarization fluorimetry. "Strong" binding was modelled by decoration of thin filaments by myosin subfragment I modified by N-ethylmaleimide (NEM-SI) or phosphorylated heavy meromyosin (pHMM). Changes in the actin structure were followed by orientation and mobility of the fluorescent probe--the rhodamine-phalloidin complex.

[A comparative study of the structural state of skeletal muscle and smooth muscle fiber tropomyosin in ghost skeletal muscle fibers by a fluorescent probe method].

The structural state of skeletal muscle and smooth muscle fiber tropomyosins in ghost fibers of skeletal muscles has been studied by means of polarization microfluorimetry. Tropomyosins and F-actin of ghost fibers were labelled with N-(iodoacetyl)-N-(1-naphthyl-5-sulfo)-ethylenediamine (1,5-IA-EDANS) or the phalloidin-rhodamine complex, respectively. It has been found that skeletal tropomyosin bound to ghost muscle fibers is more flexible in comparison with smooth muscle tropomyosin.

[The effect of the functional electrostimulation of rat fast and slow muscles on the structural state of actin in the thin filaments of a ghost muscle fiber].

The effect of electrostimulation of fast (EDL) and slow (SOL) rat muscles on the orientation and mobility of fluorescent probes rhodamine-phalloidine and 1.5-IAEDANS (N-iodoacetyl-N'-(5-sulpho-1-naphtyl)-ethylenediamine), located in various parts of actin molecule, has been studied by polarized microfluorimetry techniques. Muscles were stimulated at 20 Hz with the pulse width of 0.

[Interaction of isoforms of subfragment-1 of myosin, containing fluorescently labelled alkaline light chains, with muscle fiber actin].

Using polarization microfluorimetry, the interaction of myosin subfragment 1 (S1) isoforms containing alkali light chains A1 and A2 respectively (S1(A1) and S1(A2] with F-actin of single glycerinated rabbit skeletal muscle fibers was studied. The alkali light chains of S1 were substituted by reassociation for A1 or A2 chains modified by a fluorescent label (1.5-IAEDANS) at the single SH-group located in the C-terminus.

[The ghost muscle fiber with thin filaments reconstructed from nonmuscle actin--a model for studying the cytoskeleton using polarization microfluorimetry].

In muscle fibers which are free of myosin, tropomyosin and troponin thin filaments were reconstructed from muscle and non-muscle G-actin modified with 1,5-IAEDANS. Using polarized microfluorimetry it was shown that actin in such filaments maintained the ability to respond to conformational changes during actin interaction with subfragment of myosin (S1). The models of muscle fibers with reconstructed from non-muscle actin thin filaments are supposed to use for investigation of mechanisms of cell cytoskeleton functions with the help of polarized microfluorimetry.

[The effect of Mg-ADP on the structural state of actin in the F-actin-myosin subfragment-1 complex].

Using polarized microfluorometry techniques, a study was made on the orientation and mobility of fluorescent probes 1,5-IAEDANS and rhomadin-phalloidin, located in various parts of actin, muscle fibers free of myosin, tropomyosin and troponin (ghost fibres) being used. It was found that the binding of a myosin subfragment 1 (S1) to actin induced changes in polarized fluorescence of the fibers. The analysis of these data showed that the formation of actin-S1 and actin-S1-ADP complexes in a muscle fiber resulted in a decrease in the angle between the thin filaments and the emission dipole of phalloidin-rhodamine, as well as in an increase of the mobility of this dye.

[Effect of myosin alkali light chains on myosin subfragment 1 interaction with actin in solution and in ghost muscle fiber].

At low ionic strength (7-25 mM) Mg2(+)-ATPase of myosin subfragment 1 (S1) isoforms containing alkali light chain A1 [S1(A1)] is activated by actin 1.5-2.5 times as strongly as Mg2(+)-ATPase of S1 isoforms containing alkali light chain A2[S1(A2)].

[The effect of caldesmon and tropomyosin from smooth muscles on the motility of myosin head in ghost muscle fibers].

The effects of caldesmon and smooth muscle tropomyosin on the motility of myosin subfragment I (SI) modified by N-(iodoacetyl)-N'-(1-naphtyl-5-sulfo)-ethylenediamine (1.5-IAEDANS) was studied in myosin-, troponin- and tropomyosin-free rabbit ghost muscle fibers using the polarized microphotometry technique. It was found that the fluorescence anisotropy initiated by the 1.

[Caldesmon and myosin subfragment-1 act differently on the structural state of 1,5-IAEDANS-modified tropomyosin in ghost muscle fibers].

The effect of caldesmon (CD) and subfragment 1 of myosin (S1) on the structural state of tropomyosin (TM) modified with N-(iodoacetyl)-N-(1-naphthyl-5-sulfo)-ethylene-diamine (1.5-IAEDANS) in single myosin-free skeletal muscle fibers was studied using polarized microfluorimetry. S1 was performed from skeletal muscles of rabbits, whereas CD and TM were prepared from the smooth muscle of chicken gizzards.

[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].

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).