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G A Nevinsky

Publications and source records attributed to G A Nevinsky.

89 records · Page 5Linked to original sources
PubMed1989

DNA polymerase I (Klenow fragment): role of the structure and length of a template in enzyme recognition.

The values of Kd and Gibbs energy (delta G degrees) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo-oligonucleotides d(pC)n, d(pT)n, and d(pA)n and hetero-oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+ (NH3)2OH](pT)7 and d[(Tp2)C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357-369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+-dependent electrostatic contact and a hydrogen bond with the enzyme. The 19-20-nucleotide fragments of the template appear to interact with the protein molecule.

DNA↗
PubMed1988

Protein-nucleic acid interaction in reactions catalyzed with DNA polymerases.

The affinities of oligothymidylates and of some analogs for the template site, of a set of oligodeoxyribo- and oligoribonucleotides for the primer site, and of dNTPs and some analogs for the substrate sites of DNA polymerase I Klenow fragment and of human placenta DNA polymerase alpha were measured using them either as competitors of affinity modification or as substrates. The data obtained enable us to hypothesize that the Me2+-dependent electrostatic contact and hydrogen bond of a single internucleotide phosphate and the hydrophobic interactions of the other nucleotide units determine the formation of oligonucleotide-template site complexes. Interaction of the primer's 3'-terminal hydroxy group and of the negatively charged adjacent phosphate with the enzyme, and Watson-Crick base pairing with the template are of crucial importance for the formation of the ternary enzyme-template-primer complex. dNTP and dNMP imidazolides inactivate enzymes via an affinity modification mechanism only in the presence of the template-primer complex. dNTP affinities exceed those of dNDPs and dNMPs, the enhancement being most significant for the substrate that is complementary to the template, thus suggesting the participation of the gamma-phosphate of dNTP in the substrate selection step.

Binding, Competitive↗
PubMed1987

The efficiency of dNTP complex formation with human placenta DNA polymerase alpha as demonstrated by affinity modification.

The interaction of deoxyribonucleoside 5'-mono-, di- and triphosphates with human placenta DNA polymerase alpha was examined. Dissociation constants of enzyme complex formation with dNMP, dNDP and dNTP were determined from the data on enzyme affinity modification by imidazolide of dTMP. The basic role of the primary template-primer interaction with the enzyme in dNTP complex formation is shown. The template-dependent nucleotide interaction does not occur in the case of dNMP and dNDP in comparison with dNTP. The significant contribution of the gamma-phosphate of dNTP in this process is demonstrated.

Binding, Competitive↗
PubMed1987

Role of nucleoside components and internucleotide phosphate groups of oligodeoxyribonucleotide template in its binding to human DNA polymerase alpha.

Affinity labelling of human placenta DNA polymerase alpha (EC 2.7.7.7) with the reactive oligodeoxyribonucleotide d(pT)2pC[Pt2+(NH3)2OH](pT)7 was used for quantitative analysis of enzyme interaction with oligodeoxyribonucleotides as templates. Dissociation constants and Gibb's energy values for different oligothymidylates d(pT)nT where n = 1-14 have been evaluated by competitive experiments of these ligands with Pt2+ reagent. The data obtained prove the formation of one Me2+-dependent electrostatic contact and a hydrogen bond between the enzyme and one phosphate of these templates. One may suppose that the hydrophobic interaction of any other monomeric link of oligodeoxyribonucleotides with the enzyme template site takes place.

DNA Polymerase II↗
PubMed1978

A method for purification of peptides from hydrolysates of proteins modified by chemically active analogues of substrates containing cis-diol groups.

A simple and rapid column procedure is described for the isolation from protein hydrolysates of peptides containing covalently bound substrate analogues with cis-diol groups. The method is based on complex formation between the cis-diol groups of peptide-bound compounds and dihydroxyborylic groups of a dihydroxyborylaminoethyl cellulose column. The method is useful for isolation of peptide(s) located in or near the active centre of enzymes after their affinity labelling by chemically active analogues of natural substrates like ribonucleotides, sugars, etc.

Adenosine Triphosphate↗
PubMed1976

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by E. coli tRNAphe containing photoreactive group.

The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C.6.1.1.20) from E.coli MRE-600 with tRNAphe containing photoreative p-N3-C6H4-NHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAphe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNAphe with the enzyme, however it results in sevenfold increase of Km in the enzymatic aminoacylation of tRNAphe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [14C]-Phe-azido-tRNAphe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 xx 10(-3) M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exhibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [14C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA enzyme interaction.

Adenine Nucleotides↗
PubMedDate not supplied

Catalytic antibodies in healthy humans and patients with autoimmune and viral diseases.

Antibodies have been first characterized as proteins produced by the immune system solely for binding other molecules, called antigens, with the goal of eliciting immune response. In this classical conception, antibodies act similarly to enzymes in specific binding to different molecules but cannot catalyze their chemical conversion. However, in 1986 the first monoclonal catalytic antibodies against a chemically stable analog of the transition state of a reaction were obtained and termed abzymes (Abzs). At present, artificial monoclonal Abzs catalyzing more than 100 distinct chemical reactions have been obtained. The discovery of IgG specifically hydrolyzing intestinal vasoactive peptide in the blood serum of asthma patients stimulated studies of natural Abzs. Numerous Abzs discovered afterwards in sera of patients with various autoimmune diseases, viral disorders, or in the milk of healthy mothers, are capable of hydrolyzing proteins, DNA, RNA, polysaccharides, or nucleotides, as well as to phosphorylate proteins and lipids. The phenomenon of catalysis by auto-Abzs is more and more in research focus. In this review we summarize new data on Abzs applications in basic science, medicine and biotechnology.

Animals↗
PubMedDate not supplied

Secretory immunoglobulin A from healthy human mothers' milk catalyzes nucleic acid hydrolysis.

The human milk secretory immune system is the first line of protection for the newborn infant against various pathogens. Secretory IgA (sIgA), the typical immunoglobulin found in secretions, can fight infections through many mechanisms. Using different methods, we have shown that sIgA from the milk of healthy women possesses DNAse and RNAse activities. The catalytic center is localized in the light chain of catalytic sIgA, while the DNA-binding center is predominantly formed by its heavy chain. The enzymic properties and substrate specificity of catalytic sIgA distinguish it from other known DNases and RNases. It is reasonable to assume that the milk DNA- and RNA-hydrolyzing antibodies are capable not only of neutralizing viral and bacterial nucleic acids by binding these antigens as well as by hydrolyzing them. The DNA-hydrolyzing activity of Abs raises the possibility that these catalytic Abs may provide protective functions for the newborn through the hydrolysis of viral and bacterial nucleic acids.

Antibodies, Catalytic↗
PubMedDate not supplied

Catalytic heterogenity of polyclonal RNA-hydrolyzing IgM from sera of patients with lupus erythematosus.

Various catalytically active IgG antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies including systemic lupus erythematosus (SLE), where their presence is most probably associated with autoimmunization. Here we show for the first time that IgM from peripheral blood of patients with SLE possesses both DNase and RNase activities: these activities were also present in Fab fragments of the IgM. Both specific enzymic activities of IgM from sera of any single patient are usually 5-10 times higher than those of IgG antibodies. The same preparations of IgM hydrolyze RNA about two order of magnitude faster than DNA. The properties of the RNases of IgM and IgG distinguished them from other known pancreatic and human sera RNases. In addition, the specific activities of the RNase activity of polyclonal IgM with the polymer substrates [RNA > poly(U) > or = poly(A) >> poly(C)], the observed range of optimal pHs, of apparent Km values for substrates and of substrate specificities varied very much for different patients. The findings speak in favor of the generation of a relatively small or an extremely large pool of polyclonal catalytic IgM by the immune system of individual patients.

Antibodies, Catalytic↗
PubMedDate not supplied

Formation of the D-loop structure complexes between DNA and oligonucleotides and affinity modification of DNA by chemically reactive derivatives of oligonucleotides lead to the recombination.

INTRODUCTION: Affinity modification of DNA by chemically reactive derivatives of complementary oligonucleotides (ODNs) and antisense ODNs has shown an application for the inhibition of gene expression and the growth of viruses and parasites in high organisms. Unfortunately, the rapid advancement of antisense therapeutic approaches is not parallel to the investigation of possible consequences of antisense and gene-directed ODNs on genetic material of the cells being treated. Here we tried for the first time to estimate a possible genetic impact of antisense ODNs and their chemically reactive derivatives on the cells using bacteria and the plasmid DNA. MATERIAL AND METHODS: Recombination of direct repeats, induced by the formation of reversible complexes of plasmid DNA with complementary ODNs and after covalent binding of the alkylating derivative of the ODNs with DNA, has been investigated. For this purpose, a polylinker sequence flanked by 165 bp direct repeats was inserted within the tet gene of pBR 327. This plasmid was used to construct DNA containing AT- and GC-rich sequences placed in the central region of the polylinker. RESULTS: Transformation of E. coli cells with the plasmids (and with mixtures of the plasmids with d(pN)17 complementary to the AT- and GC-rich sequences) did not produce deletions. After modification of plasmids with alkylating derivatives of d(pN)17, the deletion of the polylinker DNA region (recombination) revealed the restoration of the tet gene function. The same effect was found at the cell transformations with the D-loop complex of the plasmids with ODNs, but the frequency of the transformants was about 1.5-2 times lower. The data obtained demonstrate that the complexes of DNA with complementary ODNs and the modification of the plasmids by reactive ODN derivatives result in induction of the recombination process and in loss of genetic material.

Base Sequence↗
PubMedDate not supplied

Effect of different drugs on the level of DNA-hydrolyzing polyclonal IgG antibodies in sera of patients with Hashimoto's thyroiditis and nontoxic nodal goiter.

BACKGROUND: DNA-hydrolyzing IgG antibodies have been detected recently in the sera of patients with several autoimmune diseases. MATERIAL AND METHODS: The relative activity of DNA-hydrolyzing IgG from the sera of patients with Hashimoto's thyroiditis and with non-toxic nodal goiter as a function of the patient's condition were measured. The effect of different drugs on the level of DNA-hydrolyzing IgG, functional activity of thyroid gland, and improvement of clinical condition of two groups of patients were analyzed. RESULTS: We demonstrate here for the first time that IgG from peripheral blood of patients with Hashimoto's thyroiditis (65%) and non-toxic nodal goiter (38%) possesses DNAse activity. The relative level-specific activity of IgGs in hydrolysis of DNA increases with the enhancement of the relative amount of antibodies against thyroglobulin and all patients with hypothyroidism (namely a reduced concentration of thyroxine and triiodothyronine and enhanced level of thyrotropic hormone) are characterized by a high level of catalytic antibodies. CONCLUSIONS: The very widely used therapy of patients with thyroxine led only to a temporary change of the hormone concentration in the blood but did not affect the level of DNA-hydrolyzing antibodies. However, treatment with an immunosuppressive drug plaquenil (7-chloro-4(beta-diethylamine-alpha-methylbutylamie)quinoline), significantly decreased the DNA-hydrolyzing activity of Abs, which correlated with enhancement of thyroid hormone concentrations, elevation of functional activity of the thyroid gland, and improvement of the clinical state of the patients.

Antibodies, Antinuclear↗