[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. 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.

[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 1.5-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.

[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. 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.

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.

Lactate dehydrogenase-induced conformational changes of F-actin in myosin-free ghost single fibres.

The changes in conformation of F-actin induced by the binding of the glycolytic enzyme lactate dehydrogenase were studied in myosin-free single ghost muscle fibres. The formation of the lactate dehydrogenase-F-actin complex was accompanied by changes in the parameters of intrinsic (tryptophan) and extrinsic (rhodaminyl-phalloin) polarized fluorescence of ghost muscle fibre F-actin. Lactate dehydrogenase stimulated actin-activated Mg2+-ATPase of myosin subfragment 1 by 30%. F-actin of ghost fibres depressed lactate dehydrogenase activity to 20% of the initial values. It is suggested that the energy-providing mechanism is coupled with that of muscle contraction through conformational changes in F-actin.

[The effect of Mg-ADP on the structural state of actin in the F-actin-myosin subfragment-1 complex]. / Vliianie Mg-ADF na strukturnoe sostoianie aktina v komplekse F-aktin--subfragment-1 miozina.

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.

[Interaction of the enzymes of cellular energy support with the F-actin of the thin filaments of muscle fiber. I. The binding of lactate dehydrogenase with F-actin induces changes in the structural state of the components of the complex]. / Vzaimodeistvie fermentov énergoobespecheniia kletki s F-aktinom tonkikh nitei myshechnogo volokna. I. Sviazyvanie laktatdegidrogenazy s F-aktinom indutsiruet izmeneniia strukturnogo sostoianiia komponentov kompleksa.

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.

[State of the contractile apparatus during the development of a pathological process in the muscles. III. The nature and sequence of the structural changes in the contractile apparatus in Zenker's necrosis]. / Izuchenie sostoianiia sokratitel'nogo apparata pri razvitii patologicheskogo protsessa v myshtsakh. III. Kharakter i posledovatel'nost' strukturnykh izmenenii sokratitel'nogo apparata pri tsenkerovskom nekroze.

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 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]. / Vliianie funktsional'noi élektrostimuliatsii bystrykh i medlennykh myshts krysy na strukturnoe sostoianie aktina v tonkikh nitiakh tenevogo myshechnogo volokna.

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.

[Polarized ultraviolet fluorescence microscopic study of the structural changes in muscle fiber contractile proteins. V. The possible nature of the conformational rearrangements in heavy meromyosin in fiber relaxation]. / Izuchenie strukturnykh izmenenii sokratitel'nykh belkov myshechnogo volokna s pomoshch'iu poliarizatsionnoi ul'trafioletovoi fluorestsentnoi mikroskopii. V. O vozmozhnom kharaktere konformatsionnykh perestroek tiazhelogo meromiozina pri rasslablenii volokna.

The mode and degree of tryptophanyl orientation relative to muscle fiber axes within hydrophobic and hydrophylic sites of myosin macromolecule in the presence of a fluorescence quencher (acrylamide, NO-3) during rigor and relaxation of glycerinated muscle fibers were studied using the polarized ultraviolet fluorescent microscopy. It was shown that myosin tryptophanyls both in LMM and HMM are oriented with their short axes along the longer axis of muscle fiber. Tryptophanyls in LMM have a more pronounced anisotropy of orientation in comparison with the fluorophore orientation anisotropy in hydrophobic sites of HMM. During the muscle fiber relaxation, conformational changes in HMM take place owing to which a section of polypeptide chain with a hydrophilic fluorophore is probably submerged deep into the macromolecule and becomes unapprochable to the quencher.

[Study of structural changes in muscle fiber contractile proteins using polarization ultraviolet fluorescent microscopy. IV. Several features of the conformational changes in F-actin during muscle fiber relaxation]. / Izuchenie strukturnykh izmenenii sokratitel'nykh belkov myshechnogo volokna s pomoshch'iu poliarizatsionnoi ul'trafioletovoi mikroskopii. IV. Nekotorye osobennosti konformatsionnykh perestroek F-aktina pri rasslablenii myshechnogo volokna.

Using the polarized ultraviolet fluorescent microscopy in the presence of a fluorescence quencher (acrylamide, NO3-) it has been shown that actin tryptophanyls are oriented with their short axes perpendicular to the long axis of the thin filament. Fluorophores in hardly accessible sites of the macromolecule have a more pronounced anisotrophy of orientation in comparison with those in the protein sites easily available by a fluorescence quencher. During the muscle fiber relaxation, conformational changes of F-actin take place which embrace seemingly the sites of subunits, close to the surface of macromolecule, and make difficult the penetration of univalent ions and neutral molecules deep into the protein macromolecule. Some connection between conformational changes of the surface areas of F-actin subunits and the actin incapability of combining with HMM in the presence of ATP is assumed.

[State of the contractile apparatus during the development of a pathological process in the muscles. V. The effect of Zenker's necrosis and of denervation atrophy on F-actin structure]. / Izuchenie sostoianiia sokratitel'noigo apparata pri razvitii patologicheskogo protsessa v myshtsakh. V. Vliianie tsenkerovskogo nekroza i denervatsionnoi atrofii na strukturu F-aktina.

By means of polarized UV fluorescent microscopy, the state of F-actin was studied in single glycerinized muscle fibers from intact, locally damaged and denervated m. semi-tendinosus of the frog. It was shown that F-actin of denervated muscle fiber lost the ability to reply by increasing tryptophan fluorescence anisotropy during the fiber relaxation and its stretching in the rigor solution by 1--4 per cent compared to the original length. Zenker's necrosis retains this ability only slightly. It is supposed that both the denervation atrophy and Zenker's necrosis change the structure of F-actin.

[Structural changes in muscle fiber contractile proteins studied by polarization ultraviolet fluorescence microscopy. VI. Conformational restructurings of F-actin induced by the binding of heavy meromyosin]. / Izuchenie strukturnykh izmenenii sokratitel'nykh belkov myshechnogo volokna s pomoshch'iu poliarizatsionnoi ul'trafioletovoi fluorestsentnoi mikroskopii. VI. Konformatsionnye perestroiki F-aktina, indutsirovannye sviazyvaniem tiazhelogo meromiozina.

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.

[Nature of the orientation of the tryptophan residues in the myosin and actin from striated muscle fiber]. / O kharaktere orientatsii triptofanovykh ostatkov v miozine i aktine poperechnopolosatogo myshechnogo volokna.

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.

[State of the contractile apparatus in the development of a pathological process in muscle. IV. Effect of Ca2+ on the process of contraction nodule formation and on the ATPase activity of muscle actomyosin in Zenker's necrosis]. / Izuchenie sostoianiia sokratitel'nogo apparata pri razvitii patologicheskogo protsess a v myshtsakh. IV. Vliianie Ca2+ na protsess formirovaniia uzlov sokrashcheniia i ATFaznuiu aktomiozina myshts pri tsenkerovskom nekroze.

Using phase-contrast and polarized ultraviolet (UV) fluorescent microscopy, the structure of single muscle fibres was studied in the course of the contraction module formation during Zenker's necrosis. The degree of manifestation of destructive changes in the contractile system was shown to depend upon the concentration of extracellular Ca-ions. With decreasing Ca2+ concentration, the fibre loses the ability to form contraction nodules peculiar to the Zenker necrosis, and the development of this process is interrupted at the stage of sarcomere supercontraction. The UV fluorescent anisotropy pattern of fibre regions, conforming with the contraction nodules, suggests the occurrence of a more pronounced disorganization of contractile system in the presence of Ca2+. The ATPase activity of actomyosin isolated from altered muscle was studied to appreciate the functional state of the contractile system. This actomyosin was found to be inactivated 1.5 times as much as that isolated from muscles treated during Zenker's necrosis in calcium-free media.

[Study of structural changes of contractile muscle proteins with the aid of polarization ultraviolet fluorescence microscopy. 1. Conformational changes of F-actin in the muscle fiber caused by ATP and its analogs]. / Izuchenie strukturnykh izmenenii sokratitel'nykh belkov myshechnogo volvokna s pomoshch'iu poliarizatsionnoi ul'trafioletovoi fluorestsentn*56i mikroskopii. 1. Konformatsionnye izmeneniia F-aktina v myshechnom volokne, vyzvannye ATF i ego analogami.

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.

[State of the contractile apparatus during the development of a pathological process in the muscles. II. The effect of Ca2+ on the morphology of spreading necrosis caused by UV light]. / Izuchenie sostoianiia sokratitel'nogo apparata pri razvitii patologicheskogo protsessa v myshtsakh. II. Vliianie Ca2+ na morfologiiu rasprostraniaiushchegosia nekroza, vyzvannogo UF svetom.

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.

[Structural changes in the contractile proteins of muscle fiber studied by polarization ultraviolet fluorescence microscopy. IX. The effect of the pH and ionic strength of the solution on the conformational restructurings of F-actin induced by the binding of heavy meromyosin]. / Izuchenie strukturnykh izmenenii sokratitel'nykh belkov myshechnogo volokna s pomoshch'iu poliarizatsionnoi ul'trafioletovoi fluorestsentnoi mikroskopii. IX. Vliianie pH i ionnoi sily rastvora na konformatsionnye perestroiki F-aktina, indutsirovannye sviazyvaniem tiazhelogo meromiozina.

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.

[Sarcomere supercontraction during Zenker's necrosis of skeletal muscle fibers]. / Sverkhsokrashchenie sarkomerov vo vremia tsenkerovskogo nekroza skeletnykh myshechnykh volokon.

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.

[Electrophoretic study of the protein makepup of the muscles in Zenker's necrosis]. / Elektroforeticheskoe issledovanie belkovogo sostava myshts pri tsenkerovskom nekroze.

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.