[Effect of continuous and impulsive ultrasound on actomyosin superprecipitation reaction from rabbit skeletal muscles].

A comparative study of the effect of continuous and impulsive (2 ms) ultrasound regimes on actomyosin superprecipitation reaction of the rabbit skeletal muscles was carried out. From the obtained kinetic curves the value of superprecipitation (D(m) - D(00)), time t1/2, which required to achieve a half its value was determined, and the normalized maximal rate of this reaction V(n) was calculated as well. It is shown that continuous ultrasound with intensities 0.7 W/cm2 and impulsive ultrasound--of 0.4 W/cm2 produced the most pronounced effect (value of superprecipitation and V(n) were maximal). The actomyosin superprecipitation value under continuous and impulsive ultrasound with intensity 1 W/cm2 relative to control and all other applied intensities was most decreased. It is caused perhaps thermal influence of the ultrasound. Thus the obtained data give every reason to assume, that impulsive ultrasound causes changes of adaptive character in actomyosin because the interrupted regime of the ultrasound signal.

[Interaction of annexin VI with actin]. / Vzaiemodiia aneksynu VI z aktynom.

Actin polymerization was investigated using fluorescence probe N-(1-pyrenyl)iodoacetamide, which was bound covalently to reactive sulfhydryl group, Cys-373. Labeled actin in the bulk was 0.5 to 1% of total actin concentration. Actin polymerization at concentration 12 mM was started by addition of 20 mM KCl and 2 mM MgCl2. The label fluorescence was excited at 365 nm and registered at 386 nm. Under actin polymerization the label fluorescence increased almost 10 times. Two main phases may be distinguished in the process of actin polymerization: 1) monomer activation and nucleus (trimer) formation, 2) growth of actin filaments on the nuclei. In our experimental conditions, both for pure actin and for that with added annexin VI, the 1st phase continued for about 3 min and after that the 2nd phase was perfectly approximated by exponential dependence. An analysis of the exponential curves showed that actin monomer lifetime increased from 327 s, at annexin absence, to about 373 s at 0.7 microM annexin and more. Calculation of rate constants at two ends of growing actin filament suggests that annexin VI binds with pointed ("slow") end so that at sufficient annexin concentration the filament grows only on barbed ("fast") end. Our results, together with data of other researchers showing that annexin VI binds with the inner membrane surface of smooth muscle cell through Ca2+, may indicate that, at Ca2+ entering the cell, this annexin binds actin filament pointed ends to cell surface making it ready for the act of contraction.

[Conformational modifications of smooth muscle light chain kinase]. / Konformatsiina modyfikatsiia kinazy lehkykh lantsiuhiv miozynu hladenkykh m'iaziv.

Our recent investigations have shown that smooth muscle myosin light chain kinase (MLCK) exists in solution as a mixture of oligomeric, dimeric and monomeric species; besides during preincubation (maintaining of the activated enzyme without substrate) with substoichiometric amounts of calmodulin (CaM) it undergoes definite changes leading to several fold lowering of its activity. Fluorescent data obtained in this work suggest that such kinase inhibition must not be connected with quantitative redistribution of different kinase species but rather it is the result of conformational modifications of this enzyme activated molecules leading to the reduction of their affinity to CaM. Such conformational rearrangements took place also at equimolar kinase to CaM ratio (or CaM excess) but in this case they were characterized by lower depth and insignificant MLCK activity fall. The nature of these conformational changes is discussed.

[Effect of regulatory light chains of myosin on aggregation of its subfragment-1]. / Vplyv rehuliatyvnykh lehkykh lantsiuhiv miozynu na ahrehatsiiu ioho subfrahmentu-1.

Chymotryptic (Ch-Cl) and Mg(2+)-papain subfragments 1 of (Mg-Cl) skeletal myosin has been studied. Mg-Cl is known to differ from Ch-Cl by the presence of the regulatory light chain (RLC) and elongated heavy chain including C-end hinge segment. Experimental data prove the decisive part of coordination bondings with bivalent cations in stabilization of RLV on myosin head hinge segment. Hydrophobic interactions are also significant that is evidenced by the intensive aggregation between subfragment Mg-Cl molecules, after RLC dissociation.

[Structural characteristics and aggregation of myosin heads in skeletal muscles]. / Strukturnye osobennosti i agregatsiia golovok miozina skeletnykh myshts.

Native conformational modifications of rabbit skeletal muscle myosin and its subfragment-1 (S-1) within the temperature range of 0-40 degrees C and irreversible unfolding of these proteins structure at temperatures 40-70 degrees C have been studied by the fluorescence and light scattering methods. The results obtained permit stating that myosin and its active subfragments form associates at the concentrations above 0.3 microM. Hydrophobic interactions between definite sites of S-1 are likely to be primarily responsible for the association. The complex profile of S-1 melting curve at high ionic strength indicates the existence of three structural domains in the heavy chain of the myosin head.

[Spectrofluorimetric detection of two states during melting of ribonuclease T1]. / Spektrofluorimetricheskoe obnaruzhenie dvukh sostoianii pri plavlenii ribonukleazy T1.

The melting temperature of ribonuclease T1 was studied by the fluorescent method. It was shown that in the melting region the tryptophanyl fluorescence spectrum of the protein containing a single tryptophanyl is the sum of two simple spectra typical for tryptophanyl located in the hydrophobic environment and for tryptophanyl completely accessible to aqueous solvent, correspondingly. This implies the evidence of two forms of the protein, i.e. native (folded) and denatured (unfolded), in the transition region. No intermediate states were found in measured quantities. Therefore, ribonuclease T1 melting process corresponds to the two states model. The free energy of native structure stabilization of the protein at room temperature is delta G approximately equal to 37 kJ/mol.