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Biomedical subjects

G A Nevinskiĭ

Publications and source records attributed to G A Nevinskiĭ.

At least 19 recordsLinked to original sources
PubMed2003

[Association between DNA antibodies levels in the blood of patients with multiple sclerosis and clinical presentation of the disease].

Multiple sclerosis (MS) as an autoimmune disease is characterized by occurrence of high titres of antibodies (AB) to cell structures and autoantigens. Various AB-mediated effector mechanisms can participate directly in the pathogenesis of demyelinisation damaging myelin, oligodendrocytes and nervous fibres. Antinuclear AB, including anti-DNA AB are an example of activation of humoral immunity in MS. Pathogenetic and clinical value of these AB is investigated insufficiently. The aim of this study was estimation of the AB titers in MS patients from two populations of Russia in relation to clinical features of MS. Results of examination of 83 patients with definite MS from Novosibirsk and Moscow (49 and 34 patients) are analyzed. Groups of comparison consisted of healthy donors and patients with SLE of the same age. Use of identical methods in the analysis of the data received in two various populations made the data objective as much as possible and revealed the strongest clinico-biochemical associations. Levels of AB to both native and denaturated DNA were studied. Comparison of several test-systems showed that the system produced by "Specialized scientific Labs" Company has the best sensitivity. In two populations of MS patients the levels of AB in plasma were similar and associated between each other, higher than in donors, but lower than in SLE patients. In both groups MS patients with secondary progressive MS had higher percent of samples of plasma with average and high levels of AB. In the Novosibirsk group associations of levels of AB with parameters of disease severety (EDSS and a number of FS scales) were seen. In the Moscow group levels of AB to DNA were significantly associated with time from the disease onset to certain level of disability (EDSS 3); both the analysis of average values and the correlation analysis showed a weak association of AB to DNA level and MS duration. The data testify that AB to DNA play a more important role in MS pathogenesis than was considered earlier. Catalytic AB as a part of anti-DNA AB may play a special role.

Adult↗
PubMed1990

[Template-primer-dependent inactivation of DNA polymerase alpha from human placenta by 2',3'-epoxyadenosine-5'-triphosphate].

Modification of the human placenta DNA polymerase alpha by 2',3'-epoxyadenosine 5'-triphosphate (eATP) was investigated. The latter binds to the protein both in absence and in presence of template-primer complex. However for inactivation of the enzyme, reagent-complementary template, primer and Me2(+)-ions are required. The inactivation is apparently due to the affinity modification of dNTP-binding site by eATP; covalent binding of the reagent off the enzyme's active site without affecting the DNA polymerase activity is also suggested. The enzyme inactivation by eATP and its protection from inactivation in the presence of dATP were used to determine Kd values of complexes of the enzyme with eATP (90 microM) and dATP (1 microM), the latter value being 13-times lower than Km for dATP (13 microM) in the polymerisation reaction. Using the dependence of the DNA polymerase inactivation by eATP on the primer concentration, Kd for enzyme-primer complexes were estimated. The Kd value for d(pA)10 (0.33 microM) was close to Km value (0.43 microM) for this primer. eATP was concluded to be a useful reagent for estimating the efficiency of the complex formation of different ligands with dNTP- and primer-binding sites of DNA polymerase.

Adenosine Triphosphate↗
PubMed1989

[Protein-nucleic acid interactions in reactions catalyzed by eukaryotic and prokaryotic DNA-polymerases].

A new method of estimation of dissociation constants for ligands and free energies of its binding based on the affinity modification of active centers in the presence of competitive ligands was developed. This method is designed for the analysis of protein-nucleic acid interactions in template systems. Deoxyoligoribonucleotides containing the reactive residue of cis-aquadihydroxydiaminoplatinum (II) and oligonucleotides ethylated at phosphate groups were used for the study of interactions of human placental DNA-polymerase alpha and the Klenow fragment of DNA-polymerase I from E. coli with templates and primers. A model was constructed which postulates the formation of a single Me2+-dependent electrostatic bond and of a hydrogen bond by one of template phosphates with the enzyme active center. Similar bonds form the basis for the enzyme interaction with the 3'-terminal phosphate group of the primer. Other monomeric units of the template are likely to interact with the enzyme by forming hydrophobic bonds. Other mononucleotide units of the primer are involved in complementary interactions with the template. The primer activity of dNMP and NMP in these systems has been demonstrated for the first time. The efficiency of dNMP, dNDP and dNTP interaction with DNA-polymerase was estimated from the affinity modification of the enzymes by dNTP and dNMP imidazolides. The key role of the template-primer interaction in the formation of the dNTP-binding site of DNA-polymerases was demonstrated. A significant contribution of dNTP gamma-phosphate to the template--dependent specific tuning of substrate dNTP was revealed.

Animals↗
PubMed1987

[Analysis of the structure and function of creatine kinase active sites using affinity modification].

Data of studies of creatine kinase from rabbit skeletal muscle (EC 2.7.3.2) by affinity labelling and affinity chromatography are reviewed. Efficiencies of these techniques are demonstrated for analysis of cooperative interactions of the enzyme's active sites, nature of non-equivalence of enzyme subunits, distances between active sites which are situated on different subunits, dynamics of enzyme-substrate interactions and usefulness of affinity labelling for localization of amino acid residues in the enzyme active sites.

Animals↗
PubMed1987

[Prokaryotic and eukaryotic DNA-polymerase. I. The role of internucleotide phosphate groups in the binding of a primer with the enzyme].

The mechanism of binding and elongation of the oligothymidylate primers in the systems of the DNA polymerase alpha from human placenta and DNA polymerase I from E. coli with the poly(dA) as a template was investigated. Both dTMP and dTTP were shown to be the minimal primers of DNA polymerase alpha, the affinity and V increasing 1.8- and 1.4-fold respectively upon lengthening the primer by each unit from dTMP to d(Tp)9T. Further elongation is accompanied by 1.3-fold affinity enhancement and a decrease in V. For the E. coli enzyme, a similar dependence of affinity of primer d(Tp)4T-d(Tp)14T was observed with the inflexion point corresponding to d(Tp)8T. The individual diastereomers of oligothymidylate ethyl esters (with p' and p'' corresponding to enantiomeric configuration) such as d[Tp'(Et)Tp]3Tp'(Et)T, d[Tp''(Et)Tp]3Tp''(Et)T, d(Tp)8Tp'(Et)T, d(Tp)8Tp''(Et)T, d(Tp)8Tp'(Et)TpT, d(Tp)8 X X Tp''(Et)TpT and completely esterified analogues d[Tp(Et)]7T, d[Tp(Et)]14T were shown to initiate the poly (dA)-dependent polymerization catalyzed by both enzymes. A sum of the obtained results provided the basis for a number of conjectures on the mode of primer and template binding to the enzyme, possible role of their preformed complex, as well as electrostatic interactions and hydrogen bonding.

DNA Polymerase I↗
PubMed1987

[Eukaryotic and prokaryotic DNA-polymerase. II. The role of internucleotide phosphate groups of a template in its binding with the enzyme].

The affinity of different ligands (phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of DNA polymerase alpha from human placenta was estimated. To this goal, dependences of rate of the enzyme inactivation by the affinity reagent d(pT)2pC[Pt2+(NH3)2OH](pT)7 on the concentration of these ligands as competitive inhibitors were determined. Minimal ligands capable to bind with the template site of DNA polymerase alpha were shown to be triethylphosphate (Kd 600 microM) and phosphate (Kd 53 microM). Ligand affinity increases by the factor 1.71 per added monomer unit from phosphate to d(pT) and then for oligothymidylates d(Tp)nT (n 1 to 14). The partial ethylation of phosphodiester groups does not change the efficiency of the oligothymidylate binding with the enzyme. However, the complete ethylation of these groups lowers affinity of the oligothymidylates to the enzyme by 7-9 times. The decrease is comparable with the change of Pt2+-decathymidylate affinity to the enzyme caused by Mn2+-ions. The data obtained led to suggestion that an electrostatic contact (most likely, Me2+-dependent) of phosphodiester group with the enzyme takes place. The type of contact is confirmed by Gibbs' energy change 1.1-1.4 kcal/mole. Formation of a hydrogen bond with the oxygen atom of P = O group of the same phosphate is also assumed (delta G =--4.4 . . .--4.5 kcal/mole). The other internucleotide phosphates and all bases of oligonucleotides form neither hydrogen bonds nor electrostatic contacts with the template binding site. Gibbs' energy changes by 0.32 kcal/mole when the template is lengthened by one unit. We suppose that this value characterizes the energy gain in the transition of oligonucleotide template from aquous medium to the hydrophobic environement of the enzyme active site. Comparison of Km values of oligothymidylates and their partially or completely ethylated analogues as templates in the reaction of DNA polymerization catalysed by DNA polymerase alpha from human placenta and Klenow's fragment of E. coli DNA polymerase I suggests a similar mechanism of template recognition by both enzymes.

DNA Polymerase I↗
PubMed1987

[Essential carboxylic groups of active sites of creatine kinase M- and M'-subunits in rabbit skeletal muscles].

The influence of carboxylic group modification with N-cyclohexyl-N'-beta-(4-methyl-morpholine)ethylcarbodiimide on the activity of creatine kinase was examined. The modification rate for M- and M'-subunits of the enzyme depends on the reagent concentration and the presence of Mg2+ ions. The process is described by linear dependences of logarithms of activity vs. time, indicating a pseudofirst order of reactions. The reagent inactivates M- and M'-subunits of the enzyme at approximately the same rate, modification being characterized by rate constants of 0.17 min-1 and K1 of 0.17 M. Mild alkaline hydrolysis (pH 9.2) of the modified enzyme leads to partial (30-60%) restoration of its activity. The addition of [14C]glycine methyl ester results in the irreversible incorporation of radioactivity into the protein. AMP and ATP gamma-(p-azido-anilide), the inhibitors acting competitively to nucleotide substrates, protect the enzyme against inactivation with the reagent, whereas creatine and creatine phosphate exert no influence on the modification rate of the enzyme. It is suggested that modification affects the aspartate or glutamate carboxyls at or near the ATP-binding sites in the M- and M'-subunits of the enzyme.

Animals↗
PubMed1987

[Creatine kinase from rabbit skeletal muscles: formation of O-acyltyrosine as a result of the activation of the carboxylic group of the enzyme active site by affinity reagents, nucleotide imidazolides].

Creatine kinase from skeletal muscle (EC 2.7.3.2) was inactivated by means of imidazolides of AMP, ADP, ATP. Rates of the inactivation of the enzyme's M- and M'-subunits differ 50-100 fold and decrease in the presence of ADP and ATP. Differential spectrum of the native and modified enzymes corresponds to the spectrum of N,O-diacetyltyrosine. Kinetic curves of hydroxylamine-dependent destruction of N,O-diacetyltyrosine and of alteration of differential spectrum of the modified and native enzymes essentially coincide. The enzyme's inactivation appears to be caused mainly by the formation of a bond between nucleotide imidazolides activated carboxyl group of the active centre and OH-group of Tyr residue arranged in the close proximity. The stoichiometry of acyltyrosine formation is evaluated as 2.1 +/- 0.2 mole per mole of the functional dimer. Along with formation of ester bond between amino acid residues, a covalent attachment of 0.03-0.06 mole of [14C]nucleotides per mole of enzyme is observed. As the data of acid hydrolysis show, Im-ATP and Im-AMP block epsilon-amino group of Lys and guanidine group of Arg, respectively. Reasons of the multiple modification of creatine kinase by affinity reagents are discussed. The results obtained and literature data are summarised in the hypothetical scheme of disposition of various amino acid residues in the active centre of creatine kinase.

Adenine Nucleotides↗
PubMed1986

[DNA-polymerase alpha from human placenta. Effectiveness of interaction between oligothymidylates of different lengths and the template-binding site].

Modification of human placenta DNA polymerase alpha by (pT)2pC[Pt2 + (NH3)2OH].(pT)7 was investigated. The linear time dependence of the enzyme activity logarithm suggested a pseudo-first order for modification. Kd value of enzyme-affinity reagent complex (0.5 microM) was estimated. The enzyme inactivation by the affinity reagent and protection from inactivation in the presence of oligonucleotides of varying length were used for determining Kd values of the enzyme-ligand complexes. Oligonucleotide d(pT)2pC(pT)7 (Kd 0.15 microM), d(Tp)9T (Kd 0.15 microM) and [d(Tp)9]ddT (Kd 0.15 microM) protected the enzyme from inactivation with equal efficiency. The protective action of oligothymidylates d(Tp)nT (where n changes from 3 to 14) strongly depended on the chain length, the Kd values diminishing from 5.3 to 0.0091 microM in the geometrical progression. The addition of one link to the oligothymidylate chain resulted in 1.71-fold increase in the oligonucleotide affinity for the enzyme specific site. Such a change corresponds to Gibbs energy change of about 0.32 kcal/mole. It is supposed that the monomer units of pentadecathymidylate (at least beginning with the third one) in d(Tp)14T-enzyme complex form neither hydrogen bonds nor electrostatic linkages with the enzyme. Kd values of oligonucleotides as templates are shown to reflect quite well the true affinity of template for the enzyme. This affinity increases in the presence of a primer. However, the ratio of the affinity for different oligonucleotides does not change in the presence or absence of a complementary primer.

Affinity Labels↗
PubMed1985

[E. coli DNA polymerase. A study of the mechanism of primer binding using oligothymidylate analogs with ethylated internucleotide phosphate groups].

The following individual diastereomers of oligothymidylate ethyl esters (the alkyl phosphodiester group is asymmetric with R or S configuration) have been prepared: d[(Tr)8Tp'(Et)T] (I), d[(Tp)8Tp''(Et)T] (II), d[(Tp)8Tp'(Et)TpT] (III), d[(Tp)8Tp'' X (Et)TpT] (IV). A totally esterified analogue d[[(Tp(Et)7]T] (V) was obtained as a diastereomeric mixture. All oligothymidylate derivatives revealed substrate activity as primers of DNA polymerase with poly(dA) as a template. The values of the maximal reaction rates were equal to 14; 2,6; 68; 24 and 0,1% for oligothymidylates (I)-(V) with respect to Vm value (100%) for (Tp)9T. Km values of oligothymidylates (I)-(V), 2,7; 2,5; 0,51; 7,2 microM, were obtained in relation to Km for d[(Tp)9T] (0,4 microM). Diastereomers (I) and (II) were not destroyed by Klenow fragment of DNA polymerase I which has only 3'----5' exonuclease activity. However, these derivatives were hydrolyzed by complete DNA polymerase I due to its 5'----3' exonuclease activity, the reaction rate being 3-10 times lower than in case of d[(Tp)9T]. The data suggest an essential contribution to the primer binding from the positive enzyme group interaction with the 3'-end negatively charged phosphate group of oligonucleotide, together with the primer complementary interaction with the template. At least two phosphodiester groups of the oligonucleotide primer are essential for the reaction of polymerization following the correct binding.

Chemical Phenomena↗
PubMed1983

[Affinity modification of creatine kinase from rabbit skeletal muscle by 2',3'-dialdehyde derivatives of ADP and ATP].

Periodate-oxidized ADP and ATP (oADP and oATP) are substrates and affinity reagents for creatine kinase from rabbit skeletal muscle. oADP and oATP modified a lysine epsilon-amino group in the nucleotide-binding site of the enzyme. Complete inactivation is observed upon binding 2 moles oADP per 1 mole of the enzyme dimer. Modification with oADP is described by a liner dependence of the log of enzyme activity on time, testifying to a pseudo-first-order of the reaction. The reaction rate constant (ki = 8.10(3) min-1) and dissociation constant for the reversible enzyme-oADP complex (Kd = 62 microM) were determined. ADP protected the enzyme from inactivation and covalent binding of the analog, whereas oADP covalently bound to the enzyme was phosphorylated by phosphocreatine. The data obtained allow to suggest that the epsilon-amino group of a lysine residue of the active site is located in close proximity to ribose of ATP and ADP forming a complex with the enzyme. This group seems essential for correct orientation of the nucleotide polyphosphate chain in the enzyme active center, but take no immediate part in the transphosphorylation process.

Adenosine Diphosphate↗
PubMed1982

[Affinity modification of rabbit skeletal muscle creatine kinase by a fluorescent analog of ATP: gamma-(azidoanalide)-1, N6-ethenoadenosine triphosphate].

A modification of active centers of creatine kinase was performed by fluorescent analog of ATP: gamma-(p-azidoanilidate)-1, N6-etheno ATP. The affinity of the analog to the enzyme and the enzyme protection against modification by substrates were investigated. The stoichiometry of the label binding to the enzyme is 2:1. The results obtained suggest that one subunit of the enzyme is modified more rapidly than the other, the modification rate difference being insignificant. The covalent binding of the analog to the enzyme results in a 4-5 fold increase of fluorescence intensity of the label, probably due to the analog conformational changes of specific interaction with the enzyme. The label bound enhances the fluorescence upon addition of the substrates. The dissociation constant of the enzyme ADP complex calculated from the titration curve is practically equal to the value estimated from experimental data on the use of the fluorescent probe 2-toluidinonaphthalene-6-sulfonate. The possibilities of application of gamma-(p-azidoanilidate)-1,N6-etheno ATP for investigation of functional and structural characteristics of the enzyme are discussed.

Adenosine Triphosphate↗