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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.

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 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+.

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.

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.

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.

The dependence of polarized fluorescence of rhodaminylphalloin specifically bound to F-actin and the tension developed by a fiber upon phosphorylation of myosin (18.5 kD) light chains as well as on the concentration of free Ca2+ was observed during the contraction of glycerinated rabbit skeletal muscle fibers. Still greater changes in the polarized fluorescence and higher values of tension were recorded for fibers with phosphorylated light chains at low (0.6 microM) Ca2+ concentrations as well as for those with dephosphorylated light chains at high (10 microM) Ca2+ concentrations. It is concluded that phosphorylation of myosin light chains modulates skeletal muscle contraction. The mechanisms of modulation involve conformational changes in F-actin.

The effect of limited proteolysis of myosin subfragment I (S1) on conformational changes in F-actin during the formation of a rigor F-actin-S1 complex was studied, using polarized microfluorimetry. Upon the decoration of thin filaments made up of rhodaminyl-phalloin modified F-actin with subfragment I, the anisotropy of fluorescence increased. Limited proteolysis of S1 at the junctions of 27 kD-70 kD and 27 kD-50 kD-20 kD fragments as well as the destruction of the 50 kD fragment by methanol had no effect on the nature of these changes. It was assumed that during the formation of the actomyosin complex the conformational changes in F-actin were induced by the 20 kD fragment. The 27 kD and 50 kD fragments seemed to exert a weak influence on action conformation within the complex.

A simple method for obtaining glycerinated muscle fibres of m. psoas of rabbit containing regulatory myosin light chains (LC2) of different levels of phosphorylation. The glycerination conditions stimulated endogenic kinase LC2 or phosphatase LC2. Glycerinated muscle fibres contained phosphorylated and dephosphorylated (levels of phosphorylation are 95 +/- 5%, and 5 +/- 5%, respectively) LC2 of myosin. To determine the level of phosphorylation the method of polyacrylamide gel electrophoresis in 8 M urea was modified.

Using polarization microfluorimetry, the effect of Ca2+ on the mode of interaction between heavy meromyosin labeled with 1.5-IAEDANS and F-actin was studied. The results obtained suggest that both the orientation and motility of myosin heads in the F-actin-heavy meromyosin complex depend on Ca2+ concentration. The experimental results are suggestive of the existence of a myosin-coupled regulatory system in skeletal muscles.