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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 0.5. Polarized fluorimetry technique and glycerinated muscle fibers were used to study effects of CaP-40 on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to CyS-707 of myosin subfragment-1 in the absence of nucleotide, and in the presence of MgADP or MgATP. We have concluded that CaP-40 binding to actin affects the strong binding of myosin to actin but has no effect on the weak binding. Thus, the influence of the CaP-40 on the formation of strong actomyosin binding forms A · M and A · M · ADP manifests itself by a decrease in the relative content of myosin cross-bridges strongly bound with actin, which probably results in a decrease in the relative content of "switch on" actin monomers in thin filaments. This suggests that, as calponin CaP-40 selects its target the phase of strong actomyosin binding binding which preceded by a phase generating power stroke.

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. Mutations in alpha-TM associated with HCM caused a shift of TM strands to the center of the thin filament and increased a range of tropomyosin motion and affinity of this protein to actin in the ATPase cycle. In contrast, mutations in alpha-TM associated with DCM shifted the protein to the periphery of the thin filament, reduced the amplitude of the TM movement and its affinity for actin. It is proposed that anomalous behavior of TM on the thin filaments in ATPase cycle may provoke the dysfunction of the cardiac muscle in patients with HCM and DCM.

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. The maximal effect was observed after 21 days of disease development. It is suggested that one of the reasons of the contractile deficit of muscle in hyperthyreosis is inhibition of the strong binding between actin and myosin during ATPase cycle.

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 1.5-IAEDANS and in the relative amount of the randomly oriented fluorophores that indicated changes in actin subdomain-1 orientation and mobility resulting from the formation of its strong binding with S1. This effect is markedly inhibited by hypothyreosis development. The maximal effect of hypothyreosis is observed after 34 days of disease development. It is suggested that the change of thyroid status in the muscle inhibits the ability of F-actin to form strong binding with myosin which is essential for force generation.

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. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding.

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. CaD inhibits this significantly. Denervation atrophy inhibits the effect of S-1 as well but does not affect the capability of CaD decreasing the formation of strong binding in actomyosin complex. It is supposed that CaD "freezes" F-actin structure in "off" state. The denervation atrophy has no effect on CaD responsibility to bind thin filaments and to switch "off" actin monomers.

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. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.

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. The results obtained have shown that during transition of the muscle fiber from the relaxed state into the rigor and during binding of actin filaments to NEM-S1, changes of polarization parameters take place, which are characteristic of formation between actin and myosin of the strong binding and of transformation of actin subunits from the "turned-off" (inactive) to the "turned-on" (active) conformation. Binding of pPDM-S1 to actin and relaxation of the muscle fiber are accompanied, on the contrary, by the changes of orientation and of the fluorescent probe mobility, which are typical of formation of the weak ("non-force-producing") form of actin-myosin binding and of transformation of actin subunits from the active conformation into the inactive one. Caldesmon and its C-terminal fragment markedly inhibit formation of the strong binding at rigor and activate transition of actin monomers to the switched off conformation at relaxation of muscle fiber. In parallel experiments, these regulatory proteins have been shown to inhibit an active force developed at the transition of a muscle fiber from relaxation to rigor. Besides, caldesmon and its fragment decrease the rate of actin filament sliding over myosin in an in vitro motility assay. Caldesmon is suggested to regulate the smooth muscle contraction in an allosterical manner. The alterations in actin conformation inhibit formation of strong binding of myosin cross bridges to actin and activate the ability of weakly bound cross bridges to switch actin monomers from the "on" to the "off" conformation.

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. CaDH1 inhibited, whereas CaDH2 activated the effect of subfragment 1. It is suggested that C-terminal part of caldesmon may modulate the transition of F-actin subunits from the "switched on" to the "switched off" state.

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. The stage AM of ATP hydrolysis cycle was simulated. For this purpose, thin filaments were decorated by myosin subfragment-1 (S1) in the absence of nucleotide. It has been shown that S1 binding to actin is accompanied by changes in orientation and mobility of the fluorescent probes. For intact filaments, the changes of these parameters indicate the formation of a strong binding between S1 and actin. Cleavage of DNA-binding loop by subtilisin markedly inhibits this effect. The cleavage of actin by subtilisin has also been shown to diminish the changes in fiber birefringence, which takes place at the formation of F-actin-S1 complex in the muscle fiber. The spatial organization of the actin DNA-binding loop is suggested to play an important role in determining the character of myosin interaction with actin in the ATP hydrolysis cycle.

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. Transition from a weak binding state of actomyosin to a strong one was accompanied by F-actin subunit rearrangements, with C- and N-terminals moving relative to the core of thin filament. Myosin light chains and 20-kDa domain of myosin head moved in the same direction as C- and N-terminal regions of actin. The flexibility of actin filaments increased, whereas that of C- and N-terminal regions decreased sharply. Actin-myosin interaction changed dramatically tropomyosin flexibility and caused displacement of the protein relative to C- and N-terminals of actin. Actin structure "freezing" by glutaraldehyde or phalloidin, actin cleavage by subtilisin, as well as actin alteration in denervational atrophy inhibited markedly the intramolecular movement and isometric tension of muscle contraction. Besides, troponin-, caldesmon-, calponin-, and myosin-systems, regulating muscle contraction, modified actomyosin rearrangements in a Ca(2+)-dependent manner. The role of the movement of polypeptide chains in contractile proteins during muscle contraction is discussed.

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. rhodamin-phalloidin complex bound specifically to actin, testified the alteration of actin structure. The results show that during the transition of muscle fibers from relaxation to rigor the flexibility of actin filaments for EDL and SOL changes differently: increases for the former and practically does not change for the latter. The analysis of heavy myosin chains points out that SOL contains 65.43 +/- 7.26% myosin heavy chains 1 (MHC 1) and 34.57 +/- 7.26% myosin heavy chains 2A (MHC 2A). In contrast, EDL has 4.57 +/- 2.56% MHC 2A and 96.43 +/- 2.56% myosin heavy chains 2B (MHC 2B). No MHC 1 were revealed in EDL. A proposal is made that the isoformal composition of myosin heavy chains defines the character of actin-myosin interaction in slow and fast mouse muscles.

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. Tropomyosin weakens the inhibiting effect of calponin. It is supposed that calponin inhibits the sliding of thin filaments in more "all or none" fashion.

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. Tropomyosin weakens the inhibitory effect of calponin and markedly increases the effect of the 38 kD fragment of caldesmon. The results indicate similarity of molecular mechanisms of the regulation of muscle contraction by calponin and the actin-binding fragment of caldesmon. It is proposed that the regulation of smooth muscle contraction by calponin and caldesmon is carried out via the inhibition of the formation of the stage AM in ATP hydrolysis cycle.

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). In contrast, phosphorylation in high ionic strength induced changes similar to those typical for the nonforce production ("weak" binding) state (phi E did not change, while sin 2 theta decreased). It is suggested that phosphorylation of LC-2 at approximately physiological ionic strength may provide fiber relaxation by switching some of the cross-bridges to the nonforce production state at the initial stage of relaxation.

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. It has been found that calponin cooperatively changes the actin conformation, the maximal conformational changes in actin thin filaments being observed at the calponin/actin molar ratio of about 1:7. The conformational changes in actin induced by NEM-SI and pHMM are typical of strong binding. Calponin inhibited this effect. It is suggested that the mechanism of calponin regulation of smooth muscle contractility is tightly coupled to the inhibition of formation of the stage limiting the rate of ATP hydrolysis by actomyosin.

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 flexibility of the thin filaments of the ghost fibers labelled in F-actin by the phalloidin-rhodamine complex and containing smooth muscle tropomyosin is higher than that of the thin filaments containing the bound skeletal muscle protein.

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 0.3 msec, some muscles were treated for 6 h during the first day, the other muscles for 6 h a day during the next 4 days before glycerinization. Then muscle fibres freed by the extraction of myosin, tropomyosin and troponin (ghost fibres) were used. It was shown that the binding of myosin subfragment 1 (S1) to actin induced the changes in polarized fluorescence of the fibres. The analysis of the obtained data showed that the formation of actomyosin complex in stimulated muscles resulted in increasing the angle between the thin filaments and the emission dipole of rhodamine-phalloidine, as well as in decreasing the mobility of this dye. In the experiments with the 1.5-IAEDANS label, the angle of the emission dipole decreased, while the label mobility increased. It was suggested that the orientation of domains in actomyosin complex changes following the electrostimulation to affect both the conformational state of F-actin in thin filaments of ghost fibres and actin-myosin interaction.

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. It was found that in S1(A1) bound to muscle fiber F-actin the mobility of the fluorescent label is lower than in S1(A2). At the same time the S1(A1) and S1(A2) interaction with F-actin induces similar changes in polarized fluorescence of rhodamine linked to falloidine which, in turn, is specifically bound to F-actin. It is concluded that the both S1 isoforms bind to F-actin and produce similar effects on the conformational state of actin filaments in muscle fibers. Local differences between S1(A1) and S1(A2) seem to be due to the interaction of the N-terminus of A1 within S1(A1) with the C-terminal region of actin.

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. In the experiments with the 1,5-IAEDANS label the angle of emission dipole increased, while the mobility of the label decreased. These changes were smaller in the presence of Mg-ADP than in its absence. It is assumed that the changes in actin monomer structure occur when a myosin head interacts with actin. These changes are expressed as those in orientation and mobility of large and small domains of actin in thin filaments. The domain orientation in actomyosin complex changes, influenced by Mg-ADP. The data obtained allow to propose the involvement of interdomain motions of some parts of actin monomer in the mechanisms of muscle contraction.

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.

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)]. Data from analytical ultracentrifugation suggest that at low ionic strength in the absence of ATP in solution S1(A1) displays a higher affinity for F-actin than S1(A2). Such a higher affinity of S1(A1) for F-actin was also demonstrated by experiments, in which the interaction of S1 isoforms fluorescently labeled by 1.5-IAEDANS with F-actin of ghost fibers (single glycerinated muscle fibers containing F-actin but devoid of myosin) was studied. Using polarization microfluorimetry, it was shown that the interaction of both S1 isoforms with ghost fiber F-actin induces similar changes in the parameters of polarized tryptophan fluorescence. At the same time the mobility of the fluorescent probe, 1.5-IAEDANS, specifically attached to the SH-group of Cys-374 in the C-terminal region of action is markedly decreased by S1(A1) and is only slightly affected by S1(A2). The data obtained suggest that S1(A1) and S1(A2) interact with the C-terminal region of the actin molecule in different ways, i.e. S1(A1) is attached more firmly than S1(A2). This may be due to the existence of contacts between the alkali light chain of A1 of S1(A1) and the C-terminal region of actin as well as to the absence of such contacts in the case of S1(A2).

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 1.5-IAEDANS-SI arrangement in the fibers is higher in the presence of tropomyosin than in its absence. Caldesmon diminishes the fluorescence anisotropy of the fibers. Data from a kinetic analysis suggest that the motility of fluorophores in the presence of tropomyosin in thin filaments is markedly decreased. Caldesmon weakens the effect of tropomyosin on the fluorescent label motility. It was supposed that caldesmon and tropomyosin initiate conformational changes in myosin heads which are accompanied by loosening or strengthening of their bonds with F-actin, respectively. Caldesmon inhibits the effect induced by tropomyosin.

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. An analysis of experimental data revealed that CD initiates and increases the motility of 1.5-IAEDANS-TM, while S1 decreases it. In the presence of CD S1 binding to actin is accompanied by significant changes in the fluorescent label motility. It is supposed that CD and S1 induce in TM conformational changes which interfere with the protein interaction with F-actin.

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.

Changes in anisotropy of tryptophan fluorescence and in birefringence of actin filaments induced by the binding to actin of heavy meromyosin (HMM), both containing DTNB light chains and devoid of them, were found in rabbit muscle fibres free of myosin, troponin, and tropomyosin. Ca2+ was shown to affect the pattern of changes in tryptophan fluorescence anisotropy and birefringence of actin filament at the moment of HMM interaction with actin, providing HMM contains DTNB light chains. Anisotropy of tryptophan fluorescence and birefringence of actin filaments rises in the absence of Ca2+ (pCa greater than or equal to 7), while in its presence (pCa less than or equal to 6) these values drop down. Furthermore, these changes become cooperative when Ca2+ concentration increases from pCa = 7 to pCa = 6. It was shown that the binding of HMM devoid of DTNB light chains to F-actin decreases tryptophan fluorescence anisotropy and birefrigence of actin filaments, regardless of Ca2+ concentration. Ca2+-dependent structural changes of F-actin induced by interaction of heads of myosin molecules with actin are assumed to be of great importance in regulation of muscle contraction of vertebrate skeletal muscles.

Using polarized ultraviolet (UV) fluorescence microscopy, it was shown that the local damage of muscle fibres causes in their morphologically unchanged parts the alternation of regions, being in different functional states referred to as "pseudocontraction" and "superrelaxation". The pattern of UV fluorescence anisotropy suggests that conformation of contractile proteins by "pseudocontraction" is similar to that at contraction, though changes in sarcomere length do not occur. The "superrelaxation" is characterized by a desorganization of myofilaments. During the spreading of Zenker's necrosis, the "pseudocontraction" is seen transferred first into "superrelaxation", and then into irreversible contracture and rigor. "The boundary of Zenker's necrosis" overlaps with the boundary of the self-propagating irreversible contracture. There is no proper boundary of Zenker's necrosis, because the destructive changes are observed over all the muscle fibre. Contraction nodules arise in the regions of "superrelaxation" and follow the changes of the contractile system, peculiar of contracture. The study of the influence of medium ionic composition on the development of Zenker's necrosis suggests that the arising and spreading of destruction are inseparably associated with the irreverrsible changes of intracellular membrane structures, and with the possibility of propogation of the damage signal by these structures along muscle fibres.

The ultrastructure and localization of supercontracted sarcomeres during the spreading (Zenker's) necrosis of twitch and tonic frog fibres and the fast- and slow-twitch rat fibres are described. The formation of supercontracted sarcomeres proceeds mainly by the sliding mechanism. Contracted bands are characterized by homogenization of myosin and actin filaments and by the disappearance of Z-band structures. In the calcium-free medium some myosin filaments pass through the whole depth of the contracted band or are seen bending and deviating backwards upon approaching the contracted band. In calcium-free solution supercontracted sarcomeres often form narrow strips (2-5 sarcomere long), crossing the fibres. The transverse elignment of sarcomeres is typical of supercontracted portion of fibres.

The dependence of F-actin conformational changes induced by the F-actin-HMM complex on pH and ionic strength was found by polarized ultraviolet fluorescence microscopy. It is discovered that pH affects sufficiently the cooperativity of F-actin structural changes, while the ionic strength affects their depth. The actomyosin complex was supposed to be at least in two structural states, differing in their orientation as well as in flexibility of F-actin monomers.

Using phase-contrast technique and electron microscopy, a study was made of morphological changes of contractile system of striated muscle fibre during the spreading necrosis caused by ultraviolet light damage. It has been shown that the degree of manifestation of destructive changes in the contractile system depends upon Ca2+-ion concentration. The ultrastructural study of the damage region, under condition of muscle fibre stretching, made it possible to reveal the initial stages of formation of this pathological process. A possible contribution of intracellular membranous structures in spreading the destructive process along the muscle fibre is discussed.

Increase of anisotropy of F-actin fluorescence of balanus and rabbit muscle fibers under the influence of ATP, AMP and pyrophosphate in EGTA presence was detected by means of the polarized ultraviolet (UV) fluorescent microscopy methods. The fluorescence anisotropy changes are assumed to be associated with the conformational changes in the actin. ATP cause more noticeable changes of actin structure, than pyrophosphate and AMP. The conformational changes in the actin of balanus and rabbit muscle fibres were similar. ATP and its analogs induced also decrease of UV fluorescence anisotropy of A-band which appears to be associated with conformational changes in myosin. It was siggested that the changes in fluorescence of anisotropy of A-bands are due to structural changes in both HMM and LMM parts of myosin molecule.

The changes in F-actin conformation in myosin-free single ghost fiber induced by the binding of heavy meromyosin (HMM) with dephosphorylated or phosphorylated light chains-2 (LC2) have been studied by measuring intrinsic tryptophan polarized fluorescence of F-actin. It has been found that at low concentrations of Ca2+ (pCa greater than or equal to 8), the binding of HMM with dephosphorylated LC2 to F-actin in ghost fibres increases, whereas the binding of HMM with phosphorylated LC2 decreases the anisotropy of polarized tryptophan fluorescence. The effect is reversed at high concentrations of Ca2+ (pCa = 5). It has been assumed that this effect of myosin light chains phosphorylation may be due to its influence on the type of myosin head binding to F-actin.

The effects of Ca2+ on conformational changes of ghost muscle fiber F-actin induced by binding of isolated rabbit skeletal muscle myosin heads (myosin subfragment 1, S1) were investigated. The changes in F-actin induced by binding of S1 to F-actin were followed by the changes in polarized tryptophan fluorescence of F-actin. It was found that the conformational changes in F-actin during the binding of S1 free of DTNB-light chains are insensitive to the changes in Ca2+ concentration. The binding of native S1 containing native DTNB-light chains induces Ca2+-sensitive conformational changes in F-actin. These changes are observed at Ca2+ concentrations of 10(-7)-10(-6) M. Such high sensitivity to Ca2+ exceeds that of S1 containing native DTNB-light chains. It was assumed that the Ca2+-sensitive conformational changes in F-actin induced by myosin head binding reflect the structural changes in thin actin filaments. These changes seem to occur in muscle fibers during the initiation and development of contraction and may be related to the control of vertebrate skeletal muscle contraction by Ca2+.

Conformational changes in F-actin, induced by glutaraldehyde or phalloidin, were found in glycerinated m. psoas rabbit fibres (ghost and reconstructed fibres). It is shown that the conformational changes of F-actin decreased the thin filament flexibility as well as weakened fibre's contractility. It is assumed that the stabilization in F-actin structure may be an important factor involved in the mechanism of muscle contraction regulation.

The effect of caldesmon on the conformational changes of F-actin caused by myosin subfragment 1 (S-1) binding was studied, using the polarized microfluorimetry method. It was demonstrated that the polarized fluorescence of rhodaminil-phalloin specifically bound to F-actin of pure actin filaments as well as of tropomyosin-containing actin filaments changes as a result of binding to S-1. The nature of these changes depends on the presence of caldesmon in the filaments. Caldesmon was supposed to modify the conformational changes in F-actin induced by S-1.

A study was made of changes in F-actin conformation occurring in a myosin-free single ghost fibre induced by the binding of glycolytic enzyme lactate dehydrogenase (LDG) to F-actin. The formation of the complex between LDG and F-actin induces changes in the parameters of the intrinsic (tryptophan) and extrinsic (rodominil--phalloin) polarized fluorescence of F-actin of the ghost muscle fibre. It is found that LDG stimulates Mg2+-ATPase of actomyosin in solution. It is assumed that the coupling of energy-providing mechanism with that of muscle contraction may be accomplished through the conformation changes in F-actin.

Dependence between the amplitude of tension, developed by glycerinated muscle fibers during rigidity, and the character of structural changes in F-actin, induced by the formation of actomyosin complex, was studied by polarized microfluorimetry and tensiometry. It is shown that during rigidity the anisotropy of intrinsic tryptophan residues as well as of rhodamine phalloidin bound to F-actin, and amplitude of tension depend on pH (6-8) and ionic strength (mu = 0.07 M-0.14 M) of solution. Greater changes in polarized fluorescence and in amplitude of tension were registered during rigidity in solutions with low ionic strength (mu = 0.07 M) and pH 8. It suggested that the amplitude of muscle fibre tension depends on the relative quantity of actin monomers, being in the "switched on" state.

Using the polarization microfluorimetry method, it was demonstrated that the increase in the degree of phosphorylation of myosin light chains (LC2) in extended single glycerinated fibers from rabbit psoas muscle changes the anisotropy of polarized fluorescence both tryptophan residue in the rod parts of the myosin molecule and the fluorescent label-N (iodoacetyl-aminoethyl)-5-naphthylamine-1-sulfonate (1,5-IAEDANS) bound to the SH1-group in myosin molecule heads. The changes in fluorescence anisotropy during LC2 phosphorylation were observed, when the measurements were performed only in the presence of 5 mM MgCl2. It was suggested that in the presence of MgCl2 the phosphorylation of LC2 associated with myosin heads changes their orientation and causes conformational shifts in the myosin filament core.

By the method of polarized ultraviolet fluorescence microscopy, effects of ATP, Ca2+, changes in pH and ionic strength of washing solution of the structural state of thick filaments in both actin-free muscle fibers of rabbit and anisotropic discs (A-discs) of glycerinated fibers of crab were studied. The dependence of tryptophan fluorescence anisotropy of thin filaments upon physico-chemical parameters (compounds) of washing solution has been found. The structural state of thick filaments was suggested to be influenced by ATP, Ca2+, changes in pH and ionic strength of washing solution.

The mode of tryptophan residue orientation in myosin and action myofilaments of the muscle fiber was studied using polarized ultraviolet (UV) fluorescent microscopy of the muscle fiber was studied using polarized ultraviolet (UV) fluorescent microscopy technique. During an elective extraction of proteine from thick and thin myofillaments changes in UV fluorescence anisotropy of muscle fibers were detected, thus suggesting that tryptophanil residues in myosin may be oriented by their own short axes mostly parallel, but in actin--perpendicular to the muscle fiber axis. The use of acrylamide, an UV fluorescence quencher, is proposed for the control of extraction electivity of proteins from muscle fibers.

Muscle fibres, free of myosin, troponin and tropomyosin, containing thin filaments reconstructed from G-actin and modified by fluorescent label 1,5-IAEDANS were used for polarized microfluorimetric studies of the effect of tropomyosin (TM) from smooth muscles, and of subfragment 1 (S1) from skeletal muscles on the structural state of F-actin. TM and S1 were shown to initiate different changes in polarized fluorescence of 1,5-IAEDANS of F-actin: TM increases, whereas S1 decreases fluorescent anisotropy. It was suggested that the structural state of F-actin may differ in the C-terminal of polypeptide chain of actin.

Changes of protein composition within necrotic areas of muscle at the late stages of Zenker's necrosis (3--5 hours after damage) have been studied using the disc-DSN-electrophoresis method. These changes are presumably associated with a disarrangement of the structure of thick and thin fillaments. The disturbance of the contractile system is accompanied by the loss of water soluble protein specific fractions.

Decrease in tryptophan fluorescence anisotropy of a single ghost muscle fibre was found at the binding of heavy meromyosin (HMM) to actin. Polarized fluorescence revealed its peak changes at a molar ratio of HMM/actin equal to 0.1. The changes observed in polarized fluorescence at F-actin-HMM interaction were found to depend on the state of HMM. The changes in anisotropy fluorescence under the same conditions were assumed to be independent of tryptophane residues in HMM, reflecting cooperative changes in F-actin conformation. Changes in the conformation of F-actin are of great importance in muscular contraction.