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E V Petushkova

Publications and source records attributed to E V Petushkova.

At least 19 recordsLinked to original sources
PubMed1991

[Characteristics of affinity modification of myosin ATPase under the action of monoaldehyde derivatives of ADP].

It was shown that the highly purified monoaldehyde derivative of ADP obtained by partial reduction of the dialdehyde derivative of ADP causes strong irreversible inhibition of the Ca-ATPase activity of myosin subfragment I, the inhibiting effect being of the affinity modification type. The addition to the reaction medium of Mg2+ (but not Ca2+) during the subfragment I interaction with the inhibitor fully prevents the inhibiting effect at all substrates used (Ca-, Mg- or K, EDTA-ATPases). Contrariwise, the subfragment I modified in the absence of Mg2+ exhibits the same degree of inhibition for all the three types of the ATPase activity. An unexpected result that was previously unobserved for other affinity modifiers of myosin ATPase is the maintenance of activity in 50% of active centers, when "two-head" forms of the enzyme (the myosin proper and heavy meromyosin, HMM) are modified. Noteworthy that the affinity modification reaction is characterized by the same values of inhibition constants as in the case of myosin subfragment I (Ki = 3.3-3.5 X 10(-4) M; ki = 0.03-0.04 min-1). This finding provides additional evidence in favour of functional asymmetry of myosin heads in the myosin molecule which seems to be due to the screening of the active center of one head by the other one.

Adenosine Diphosphate↗
PubMed1989

[Affinity modification of myosin with protected active centers: confirmation of the existence of an allosteric substrate-binding segment].

The effect of an affinity modifier of myosin ATPase representing a mixed anhydride of AMP and mesitylene carboxylic acid (AMP-MA) on myosin with protected active centers was studied. The protection of active centers was performed by the method of Wells et al. Which consists in the stabilization of the myosin-MgADP complex in the enzyme active center by way of cross-linking of the active center with a Co-phenanthroline complex simultaneously interacting with two SH-groups of the protein. Myosin with protected active center completely loses its ability to hydrolyze ATP; however, it can be reactivated by way of SH-group reduction with a subsequent MgADP release from the active centers. Treatment of myosin with protected active centers with AMP-MA does not result in the reduction of the enzyme activity after removal of the Co-phenanthroline complex. This suggests that the irreversible inhibition of myosin ATPase by AMP-MA occurs due to the protein modification outside the active center(s), which provides support for our earlier made conclusion concerning the existence of an additional (with respect to active centers) substrate-binding site in the myosin molecule.

Adenosine Triphosphatases↗
PubMed1988

[Increased substrate selectivity during transition from Ca2+-activated to K+,EDTA-activated nucleoside triphosphatase activity of heavy meromyosin].

A comparison of kinetic parameters (Km(app) and V) of hydrolysis by heavy meromyosin of natural (ATP and ITP) and modified nucleoside triphosphates showed that in the K+, EDTA-ATPase conformation the enzyme exhibited a higher selectivity towards the structure of the substrate nucleoside moiety than in the case of the Ca2+-stimulated nucleoside triphosphatase activity. In the presence of Ca2+, all the N1- and N6-substituted analogs of ATP as well as ITP, etheno-ATP and the dialdehyde derivative of ATP were hydrolyzed at a high rate irrespective of their markedly decreased affinity for heavy meromyosin. In the presence of K+, EDTA the ATPase activity showed a tendency for a total decrease of the analog affinity for nucleoside triphosphates, i.e., the impossibility of tight binding of the substrate phosphate residues to the protein in the absence of bivalent cations, which was concomitant with an increase in the hydrolysis rate. However, it was found that only in N1-substituted analogs any appreciable changes in the substrate properties were absent. All the other nucleoside triphosphates tested (N6-carboxy-methoxy-ATP, N6-(N'-acetylaminoethoxy)-ATP, etheno-ATP, ITP and the dialdehyde derivative of ATP having a rupture in the ribose ring) lost their ability to be hydrolyzed by heavy meromyosin. The experimental results as well as the literature data are suggestive of differences in the spatial structure of the active center in two different myosin conformations associated with a high catalytic activity, i.e., K+, EDTA-ATPase and Ca2+-ATPase.

Adenosine Triphosphatases↗
PubMed1985

[Dialdehyde derivatives of purine mononucleotides: substrate properties and affinity modification of myosin ATPase].

It was demonstrated that the dialdehyde derivative of ATP is a good substrate for Ca-ATPase of heavy meromyosin (Km = (1.2-1.4) X 10(-4) M; V = VATP). At the same time, this compound can induce irreversible inhibition of the enzyme. Since oxo-ATP is rapidly hydrolyzed by myosin to form oxo-ADP, this inhibition is the result of the enzyme interaction with oxo-ADP. It was found that the kinetics of heavy meromyosin inhibition by oxo-ADP are typical of affinity modification; in this case ATP fully protects heavy meromyosin from the activity loss. Similar results on the irreversible inhibition of the ATPase activity under the action of oxo-ADP were obtained in the presence of myosin, heavy meromyosin, subfragment I and natural actomyosin and in the absence of bivalent cations, thus suggesting the modification of the active center of myosin ATPase.

Adenosine Diphosphate↗
PubMed1985

Investigation of myosin substrate-binding site using phosphorylating analogs of the substrate.

Affinity modification of HMM has been performed, using mixed anhydrides of AMP, epsilon AMP, ADP or IMP and sterically hindered aromatic carbonic acids. The affinity labelling site of HMM was demonstrated to be highly specific towards the adenosine fragment of the affinity analog. The number of phosphate groups and the hydrophobicity of the aromatic acid substitutents did not influence the mode of the analogs interaction with HMM. The data obtained confirms our previous suggestion that the nucleotide analogs in question modify the substrate binding site which is other than the active site of the enzyme.

Adenine Nucleotides↗
PubMed1984

[Several peculiarities of the purine-base fixation of the substrate in the active sites of myosin Ca2+-ATPase].

A comparative study of kinetic parameters (Km and V) of hydrolysis by heavy meromyosin of several synthetic ATP analogs with substituents at positions N(1) and N(C6) of the purine ring was carried out. Analysis of changes in the Km values suggests that the purine base of ATP is fixed in the active center due to the formation of a hydrogen bond between N1 and the proton donor group of the protein as well as between the 6-NH2-amino group of the nucleotide and the proton acceptor group of the protein. It was shown that the rate of catalytic conversion of the substrate is determined by the mode of binding of its purine ring. Depending on the properties of the substituent radical, the latter either prevents the binding by causing little or no increase in the rate of hydrolysis or causes the displacement of the whole substrate molecule in the active center, which leads to the deceleration of hydrolysis.

Adenosine Triphosphate↗
PubMed1982

[Affinity modification of heavy meromyosin and subfragment 1 by mixed anhydrides of [14C] AMP, epsilon AMP and mesitylene carboxylic acid].

Using mixed anhydride of AMP and mesitylene carboxylic acid carrying a fluorescent or radioactive label, it was found that the previously established irreversible inhibition of myosin ATPase is a result of protein covalent binding to the nucleotide residue of the inhibitor. The stoichiometry of the affinity labelling of heavy meromyosin is 1 mole of nucleotide residue of mixed anhydride per 1 mole of protein, that of subfragment 1-0.5 mole per 1 mole of protein. The lack of irreversible inhibition of the ATPase activity of subfragment 1 is suggestive of an existence of a regulatory substrate-binding site in the myosin molecule.

Adenosine Monophosphate↗
PubMed1982

[Characterization of two types of binding sites of substrate-like inhibitors in the heavy meromyosin molecule].

The effects of several phosphorylating and alkylating analogs of the substrate on the ATPase activity of myosin and heavy meromyosin were compared. The data obtained confirmed the previously made assumption on the existence of two types of substrate-like inhibitor binding sites in the enzyme molecule. In one of the sites, presumably in the active one, there occurs a reversible competitive inhibition characterized by a high affinity for the inhibitors, which are mixed anhydrides of various mononucleotides and mesitylcarboxylic acid or its derivatives. An enhancement of hydrophobicity of these compounds causes an increase in their affinity for this site. At much higher concentrations of the inhibitors an irreversible inhibition takes place, the rate of inhibition being decreased with an increase in the phosphorylating capacity of the compound. This site possesses a far lower affinity for the inhibitors and reveals a certain specificity with respect to the analog mononucleotide moiety structure, i.e. a replacement of the 6-NH2-group by the 6-OH-group or an increase in the number of the phosphate residues result in a decrease of the efficiency of inhibition. No correlation between the analog capacity to cause irreversible inhibition and to act as an effective competitive inhibitor of reversible type has been shown to exist, thus allowing to use inhibitors of preferable action in one of the two types of the binding sites. No irreversible inhibition site was revealed when the ATPase activity of myosin subfragment I with and without the DTNB chains was investigated. Actin protects myosin against the inhibiting action of the analogs tested.

Adenosine Triphosphatases↗
PubMed1980

[Inhibition of the Ca-ATPase activity of heavy meromyosin by phosphorylating analogs of the substrate].

Mixed anhydrids of AMP, ADP, ATP and IMP and mesitylene carboxylic acid (AMP-MC, ADP-MC, ATP-MC and IMP-MC) are efficient irreversible inhibitors of the Ca-ATPase activity of myosin and heavy meromyosin. The highest rate of inhibition is observed in the case of AMP-MC: at AMP-MC concentration of 1,5.10(-3) M the half inactivation time for heavy meromyosin varies in different protein preparations from 10 to 20 min. The rates of inhibition in the presence of ADP-MC and ATP-MC are roughly the same and are far lower than those for AMP-MC (half inactivation time is 1,5-2 hrs). However, in the latter case the inhibition is complete, the time of the analogs interaction with the protein being increased up to several hours. In the presence of IMP-MC the inhibition is also time-dependent but is never complete. A necessary condition for the manifestation of irreversible inhibition of the Ca-ATPase activity of TMM by phosphorylating analogs of the substrate is the presence of bivalent cations. No inhibition occurs in the presence of EDTA. An addition of ADP or ATP to the preincubation medium causes a sharp decrease of the inhibition rate (a protective effect), which suggests a specific interaction of the analogs with TMM at the substrate binding site.

Adenine Nucleotides↗
PubMed1977

[pH-dependence characteristics of Ca-ATPase activity of heavy meromyosin with modified SH-groups].

Study of pH-dependence of Ca-ATPase activity of heavy meromyosin (HMM) at low and high ionic strength showed essential differences in the modifying effect of two sulfhydryl reagents, p-CMB and silver. Silver ions in conditions studied independently on pH and KCl concentration produce an inhibition of ATP hydrolysis by myosin and HMM, the shape of the pH-dependence curve remaining similar to that of the native enzyme up to 40% of blocking free sulfhydryl groups. At the same degree of binding of sulfhydryl groups with p-CMB at 0,5 M KCl the pH-dependence curve due to activation at neutral pH changes it's shape and becomes similar to that for dissociation of two ionizable groups (at neutral and alkaline regions). In contrast to this, a low or zero concentrations of KCl no activation was observed for the enzyme with 40-50% of SH-Groups modified by p-CMB and Ca-ATPase in this case seemed to be independent of pH. The data obtained suggest that SH-Groups are not included into the active site of myosin, and the activating effect observed for some sulfhydryl reagents, is due to conformational changes and it can be the result of the penetrance of the organic part of the reagent molecule into hydrophobic region of the protein.

Adenosine Triphosphatases↗
PubMed1977

[Kinetic study of the pH-dependence of maximal rate of Ca-ATP hydrolysis by myosin].

Curves of V pH-dependence for Ca ATPase of myosin and heavy meromyosin are demonstrated to be well modelled with theoretical curves for the case of proton dissociation at three groups of enzyme-substrate complex with the loss of the activity at some intermediate ionization stage. Variation of pK values for these three groups and the degree of inhibition for intermediate forms of enzyme-substrate complex are found to be sufficient to reproduce main varieties of described in the literature and obtained in this work multiformity of pH-dependence curves of different nucleoside triphosphates hydrolysis by both native and modified enzymes. Calculated pK values and modification data suggest a significant importance of the dissociation of two imidazole groups ("activating" and "inhibitory") and cisteine sulhydryl group for the catalytic activity of myosin. Inhibition of ATPase activity by increasing of KCl concentrations is found to be due first of all to a shift in pK values of "inhibitory" imidazole and sulhydryl groups.

Adenosine Triphosphatases↗
PubMed1976

[Effect of storage conditions on the kinetic properties of myosin ATPase].

"Substrate inhibition", which has been described earlier for myosin Ca-ATPase in low ionic strength KCl solution [1], is found to take place also at high KCl concentration and under partial modification of enzyme thiol groups with p-CMB. "Substrate inhibition" disappeared when increasing Ca2+ concentration up to 25-40 mM. These kinetic properties are characteristic for fresh isolated enzyme and myosin preparations stored in 0.5 M KCl. They may change under storage of enzyme preparations at higher KCl concentrations: no "substrate inhibition" is observed after 6-8-day storage of myosin preparations in 3 M KCl at the presence of 4-5 mM CaCl2. The data on optical rotation dispersion and analytical ultracentrifugation have shown that the storage of myosin in 3 M KCl is accompanied by structural changes of the protein.

Adenosine Triphosphatases↗
PubMed1976

[Kinetic analysis of hydrolysis of free ATP and MgATP by natural actomyosin].

Computer analysis of experimental data published in 1-3 allowed to establish the presence of two non-interacting inequivalent hydrolytic sites in actomyosin molecule, one of them being specific for binding and hydrolysis of free ATP, the other--for MgATP. Thus both species of ATP are the substrates of actomyosin ATPase. Actomyosin molecule seems to bind on more (in additon to two active sites) substrate molecule (MgATP) at some non-catalytic regulatory site. The formation of the enzyme-substrate complex having three ATP molecules (one molecule of free ATP and two--of MgATP) is accompanied by the loss of the activity. An approach to the research of kinetic equations for complex systems considerably decreasing a number of variations to consider is given in this work.

Actomyosin↗