[Changes in structure and functional properties of cytoskeletal elastic protein titin in dilated cardiomyopathy]. / Izmenenie struktury i funktsional'nykh svoistv tsitoskeletnogo élastichnogo belka taitina pri dilatatsionnoi kardiomiopatii.

To elucidate the role of titin in the onset and development of dilated cardiomyopathy, the structure and functional properties of this protein from pathological myocardium (human left ventricle) were studied. By the use of SDS gel electrophoresis, a decrease in molecular weight of titin in dilated cardiomyopathy compared with norm (pig left ventricle) was revealed. The decrease correlated with the stage of the disease. A decrease in the length of molecules of pathological forms of titin was also found by electron microscopy, which confirms the results of electrophoresis tests. It was shown that, unlike titin from healthy muscle, pathological forms of titin do not activate but inhibit the main functional properties of control myosin: the actin-activated ATPase activity and its Ca2+ sensitivity. The direction of the changes in structure and functional properties of titin allows one to conclude about its contribution to the development of the pathology studied.

[Correlation between Ca2+-dependent movement of crosslinks in myosin filaments and Ca2+-sensitive actin-activated ATPase of skeletal muscle myosin]. / Korreliatsiia mezhdu Ca2+-zavisimym dvizheniem mostikov v miozinovykh nitiakh i Ca2+-chuvstvitel'nost'iu aktin-aktiviruemoi ATFazy miozina skeletnykh myshts.

The dependence of actin-activated ATPase activity and myosin filament structure have been studied on Ca(2+)-concentration in the range between pCa 7.5 and pCa 4.6. Rabbit skeletal muscle myosin with dephosphorylated regulatory light chains column-purified with DEAE-Sephadex A-50 for the removal of minor proteins and actin without regulatory proteins have been used. Considerable increase in actomyosin ATPase activity (by 70%) is revealed with increasing Ca(2+)-level from pCa 7.5 up to pCa 4.6. Electron microscopic observations on the structures of reconstituted myosin filaments have revealed Ca(2+)-dependent movement of myosin cross-bridges (head + subfragment-2) from and to the backbone of myosin filaments. The correlation between the manifestation of Ca(2+)-sensitivity of ATPase properties of myosins and Ca(2+)-dependent mobility of cross-bridges has been established. In particular, the increase in the mobility of cross-bridges and their moving away from the surface of myosin filaments at pCa 4.6 correlates with the increase in actin-activated ATPase of the same myosin preparations. It is supposed that the interrelation between the above properties observed in in vitro system can be of importance for force generation and its regulation in muscle.

[Features of the effect of ammonium ion on the enzymatic activity of myosin and subfragment-1]. / Osobennosti vliianiia iona ammoniia na fermentativnuiu aktivnost' miozina i subfragmenta-1.

The kinetic isotope effect of hydrolysis of ATP by myosin subfragment-I in the presence of K+, NH4+, Rb+ was measured. VH/VD was found to be 1.8; 1.3; 2.0, respectively. According to the thermodynamic isotope effect induced by hydration, the kinetic isotope effect must increase with the increase of cation size from K+ to Rb+. The size of ammonium ions is the intermediate between K+ and Rb+, but the observed isotope effect in the presence of ammonium is much lower than in the presence of K+ and Rb+. The results suggest that monovalent cations occur near the active center of the enzyme and contribute to some extent to the hydrolysis reaction but the specificity of ammonium ions seems not to be due to its ideal steric accordance. The obtained results support the viewpoint that NH4+ ions as donor of protons participate in the chemical stage of ATP hydrolysis by subfragment-I.

[Changes in mechanical characteristics of immobilized crystals and films of globular proteins during substitution of D2O for H2O]. / Izmenenie mekhanicheskikh kharakteristik immobilizovannykh kristallov i plenok globuliarnykh belkov pri zamene H2O na D2O.

The influence of substitution of the isotopic composition of the medium on the mechanical properties of immobilized crystals and films from bovine pancreatic ribonuclease and hen egg white lysozyme was investigated. The order of magnitude of the observed effects indicates that the contribution of the electrostatic interaction to the observed isotopic effect may be considered inessential. The absence of aggregation in the H2O and D2O medium under experimental conditions is demonstrated by the method of the low angle dispersion of X-rays. The observed effects of D2O on the mechanical behavior of crystals and films of proteins may be accounted for by the strengthening of molecular interactions in the samples.

[Analysis of kinetic isotope effects in H2O-D2O mixtures in enzymatic catalysis: detection of conformation-dynamic processes]. / Analiz kineticheskikh izotopnykh éffektov v smesiakh H2O-D20 pri fermentativnom katalize: vyiavlenie konformatsionno-dinamicheskikh protsessov.

An analysis was carried out of Gross-Butler type equations describing relationship between deuterium isotope effect in reactions with proton transfer and deuterium concentration in the medium. It has been shown that with all possible coefficient values of isotopic fractionation of enzyme functional group protons the shape of indicated relationships qualitatively differs from the experimentally observed one for myosin hydrolysis of ATP. This discrepancy as well as uniform nonlinear change of myosin denaturation temperature and of kinetic isotope effect in myosin hydrolysis of ATP on deuterium fraction in solution give evidence of D2O effect as a solvent and point to an essential role of conformational - dynamic processes in the course of enzymic catalysis with myosin.

[Possible role of structural changes in ATP hydrolysis by subfragment-1 and myosin]. / O vozmozhnoi roli fluktuatsii struktury pri gidrolize ATF subfragmentom-1 i miozinom.

Dependence of the rate of ATP hydrolysis with subfragment-I and temperature of SF-I, denaturation on the concentration of heavy water in solution was studied. The value of kinetic isotope effect V/Vx linearly increases with the rise of the volume portion of heavy water in solution and at X-1 it equals 1.9. The temperature of protein denaturacticn increases with X rise, the pattern of this relationship looking as an arched curve. The results differ from those earlier obtained on myosin which points to the absence of essential contribution of structural dynamic changes to enzymic hydrolysis of ATP by subfragment-I.

[Enzyme activity of myosin activated by different cations in a mixed H2O--D2O solvent]. / Fermentativnaia aktivnost'miozina, aktiviruemaia razlichnymi kationami v smeshannom H2O--D2O rastvoritele.

The rate of enzymic reaction of ATP, ITP, GTP with myosin is studied in the presence of potassiu, ammonium and calcium ions in H2O--D2O solutions. There is no kinetic isotope effect of ITPase and GTPase reaction in the neutral pH region (VHVD = 1). The value VH/VD for the ATPase reaction in the pH range from 6.5 to 8.5 with all cations studied varies from 1.05 to 1.26. Such changes of myosin enzymic activity in D2O infer that small changes in the interaction of subunits is not the decisive one in the regulation of myosin ATPase. The equality of isotope effects in potassium salts and ammonium solution suggests that a specific effect of ammonium ion as a proton donor affects the ATPase reaction of myosin. The relationship between the value of isotope effect and D2O concentration in solution in non-linear. The shape of concentration curve suggests essential conformational changes of myosin during ATP hydrolysis.

[Comparative study of myosin and DTNB-treated myosin with regard to ATP activity and fluorescence]. / Sravnitel'noe issledovanie miozina i DTNB-obrabotannogo miozina po ATPaznoi aktivnosti i fluorestsentsii

When studying enzymic and fluorescence properties of myosin and DTNB-treated myosin in the presence of K+, Na+, Li+, NH4+, Ca2+ and Mg2+ cations the following results were obtained. By the intrinsic protein fluorescence techniques no essential structural changes of myosin molecule at the dissociation of the DTNB light chain and activation myosin ATPase in the presence of different cations were found. The decrease of K+-EDTA-, the increase of Mg2+-activated and the stability of Ca2+-activated myosin ATPase may be the result of the modification of SH1 or SH2 sulfhydryl groups when treating the DTNB myosin in our conditions. The different level of decrease of the K+- and NH4+-activated myosin. ATPase may be explained by the fact, that myosin sulfhydryl groups have different effects on the activation of its ATPase by these cations.

[Study of carnosine complexes with nucleotides by the method of high resolution nuclear magnetic resonance]. / Issledovanie kompleksov karnozina s nukleotidami metodom iadernogo magnitnogo rezonansa vysokogo razresheniia

It is shown that the formation of a carnosine--nucleotide complex (ATP, ADP, AMP) takes place. The stability of the complex mainly depends on: 1) the staking interaction between the heterocyclic rings of carnosine and nucleotides; 2) the electrostatic interaction between the phosphate groups of nucleotide and the positive charged amino group NH3+ of the beta-alanine part of carnosine. The formation of the hydrogen bond between dipeptide COO- group and N1 or N7 of nucleotide is also possible. The complex stability strongly depends on the charge-state of the components and little on the number of the phosphate groups of nucleotide (ATP greater than or equal to ADP greater than AMP).