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Biomedical subjects

G A Nevinsky

Publications and source records attributed to G A Nevinsky.

At least 55 records · Page 3Linked to original sources
PubMed1998

Phosphorylation of lipids tightly bound to secretory immunoglobulin A in antibody fractions from human breast milk possessing protein kinase activity.

The fractions of antibodies (Abs) containing only sIgA and IgG were purified from human breast milk by Protein A-Sepharose chromatography and they catalyzed phosphorylation of casein in the presence of [gamma-32P]ATP. Also, 32P-labeled low-molecular-weight non-protein products are formed which are visible as radioactive background on the polyacrylamide gel lanes during electrophoresis of Abs under denaturing conditions. Separation of sIgA from IgG using a DEAE-sorbent with subsequent gel-filtration in 0.05 M NaOH indicates that the low-molecular-weight substances partially remain tightly bound to sIgA and are separated only by gel-filtration in a buffer containing 5% dioxane (non-denaturing resolution) or by extraction of the sIgA pellet with the chloroform--methanol mixture (2:1). The 32P-labeled substances were separated by TLC in the system used for phospholipid chromatography forming two fractions (Rf 0.83 and 0.66) that were stained with iodine. The data suggest that the substances co-isolated with sIgA are phospholipids. At 25 nM ATP, casein and lipids are 32P-labeled. At 1 microM ATP, the sIgA polypeptides are also phosphorylated. Gentle removal of the lipids from Ab preparation enhanced 32P incorporation into casein and sIgA polypeptides. Considering the heterogeneity of polyclonal sIgA in protein and ATP affinity, it is suggested that phosphorylation of casein and sIgA polypeptides is catalyzed by abzymes of different clonal origin.

Chromatography, Liquid↗
PubMed1998

Human breast milk immunoglobulins G hydrolyze nucleotides.

Catalytically active antibodies, abzymes, appear in the blood of mammals immunized with the analogs of transition state or in the case of autoimmune diseases. Until recently, it was not shown whether abzymes exist in the blood of apparently healthy subjects. We have discovered that secretory IgA (sIgA) from the milk of healthy mothers catalyze phosphorylation of a variety of proteins and that IgG can hydrolyze DNA and RNA. In this study for the first time it is shown that IgG from human milk (and their Fab-fragments) also catalyze hydrolysis of nucleoside mono-, di-, and triphosphates. The data meet certain stringent criteria, unambiguously indicating that the observed catalytic activity is associated with IgG rather than contaminating enzymes. Although the nucleotide-binding site of IgG is located in the light antibody chain, L-chains per se cannot hydrolyze NTP unlike the DNA-hydrolyzing abzymes. Kinetic and thermodynamic parameters that characterize the interaction of NTP and dNTP with IgG-abzymes were analyzed. Possible reasons for appearance of polyclonal abzymes with different catalytic activities in the milk of healthy mothers are considered.

Adenosine Triphosphate↗
PubMed1998

Interaction of human milk lactoferrin with ATP.

Human lactoferrin exhibits many unique properties. It is known as one of the most important factors that provide nonspecific defense of cells against bacteria, viruses, and carcinogenesis, as well as an important component of a specific system responsible for the passive immunity of newborns. As a compound with extremely broad spectrum of functions many of which were not elucidated so far, lactoferrin is intensely studied. In this study we obtained electrophoretically and immunologically homogenous preparations of lactoferrin from human milk. Using various methods, we were the first to show that the fraction of lactoferrin, which displays an increased affinity for Sepharose Blue, forms complexes with ATP with a stoichiometry of 1 mole ATP per mole protein. It is shown that the ATP-binding site is located in the C-terminal domain of the lactoferrin molecule. The binding of ATP results in the dissociation of tetrameric forms of the protein and a change in the mode of interaction of lactoferrin with polysaccharides and other proteins. The data may be used in analysis of the possible reasons for multifunctional properties of lactoferrin and possible ways of regulation of its functions.

Adenosine Triphosphate↗
PubMed1998

Polyclonal antibodies from blood and cerebrospinal fluid of patients with multiple sclerosis effectively hydrolyze DNA and RNA.

It is known that in the blood of patients with some autoimmune diseases catalytically active antibodies hydrolyzing proteins, DNA, and RNA may be detected. In the present work homogeneous preparations of IgG antibodies (Ab) possessing high affinity for nucleic acids (NA) were obtained for the first time from blood and cerebrospinal fluid of patients with multiple sclerosis (MS). The fraction of IgG Ab as well as its Fab fragments and isolated light chains of both kappa- and lambda-types were shown to catalyze effectively the hydrolysis of DNA and RNA. It is shown by different methods that the capability for nucleic acid hydrolysis is an intrinsic property of the polyclonal Ab. NA-hydrolyzing Ab were detected in the blood of 69 of 72 and in the cerebrospinal fluid of 5 of 5 examined MS patients, while they were not detected in the blood of any of 50 healthy donors examined. Comparison of relative rates of RNA hydrolysis and of the substrate specificity in hydrolysis of various model RNAs--cCMP, poly(U), poly(A), and poly(C)--revealed pronounced differences of MS antibodies from ribonucleases of human blood, ribonuclease A, and all earlier described abzymes. The abzymes are usually characterized by relatively low specific activities in comparison with that of normal enzymes catalyzing analogous reactions. Ab from the blood of MS patients are the first example of autoabzymes whose specific activity in RNA hydrolysis is comparable or even higher than that of pancreatic ribonuclease A--one of the most active RNA-hydrolyzing enzymes.

Antibodies, Catalytic↗
PubMed1997

DNA-hydrolyzing activity of the light chain of IgG antibodies from milk of healthy human mothers.

Various catalytically active antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Normal humans are generally considered to have no abzymes, since no obvious immunizing factors are present. Recently we have shown that IgG (its Fab and F(ab)2 fragments) from the milk of normal humans possesses DNase activity. Here we demonstrate for the first time that the light chain of IgG catalyzes the reaction of DNA hydrolysis. These findings speak in favor of the generation of abzymes in the tissue of healthy mothers, and since a mother's breast milk protects her infant from infections until the immune system is developed, they raise the possibility that these abzymes may contribute to this protective role.

Antibodies, Catalytic↗
PubMed1997

High affinity interaction of mouse DNA topoisomerase I with di- and trinucleotides corresponding to specific sequences of supercoiled DNA cleaved chain.

Recently mouse DNA topoisomerase I (topo) was shown to possess high affinity for a single-stranded AAGACTTAG nonanucleotide (K(i) = 2.0 microM) corresponding to the scissile strand of the minimal DNA duplex, which is necessary for cleavage of supercoiled DNA. In order to determine the most important part of the above sequence for the DNA recognition by topo, the interactions of the enzyme with a set of extremely short (2-5 nucleotides in length) oligonucleotides corresponding to different parts of the nonanucleotide have been investigated. The affinities of different oligonucleotides corresponding to the CTTAG part of the sequence (K(i) = 0.13-0.92 mM) were shown to be significantly lower than that for the AAGA tetranucleotide (K(i) = 9.0 microM). Topo effectively recognized even short oligonucleotides containing only two or three bases (AGA and pAG, K(i) = 20 and 50 microM). We suppose that oligonucleotides having a high afffinity to the enzyme can offer a unique opportunity for the rational design of topoisomerase-targeting drugs.

Animals↗
PubMed1997

Recognition and conversion of single- and double-stranded oligonucleotide substrates by 8-oxoguanine-DNA glycosylase from Escherichia coli.

The substrate specificity of 8-oxoguanine-DNA glycosylase was studied. The data showed for the first time that the enzyme can cleave single-stranded deoxyoligonucleotides containing an 8-oxoG link. The values of K(m) and Vmax for a range of single-stranded and double-stranded oligonucleotides (23 bp) containing 8-oxoG at different positions of one chain. These parameters consistently depended on the position of 8-oxoG in single-stranded or double-stranded substrates. A possible mechanism responsible for substrate recognition by 8-oxoguanine-DNA glycosylase is described.

Base Sequence↗
PubMed1997

RNA-hydrolyzing antibodies from peripheral blood of patients with lupus erythematosus.

Experiments and hydrolysis of substrates with known spatial structures (such as yeast tRNAPhe, as well as normal and mutant tRNALys from human mitochondria produced by transcription of the appropriate DNA species, that is, RNA genes) were performed to study the ribonuclease activity of antibodies isolated from blood sera of patients with systemic lupus erythematosus (SLE). The antibody preparations contained two types of ribonuclease activities: the first corresponded to the specificity of ribonuclease A and was found during hydrolysis at low salt concentrations, whereas the second was stimulated by Mg2+ and displayed unique specificity toward double-stranded regions of the substrate. The possible use of the antibody preparations as tools for structural studies of conformational differences between RNA molecules was examined. In experiments with unmodified and mutant tRNALys species differing in one base found in the T-loop, we found that hydrolysis with SLE antibodies can detect small local structural changes in RNA under physiological conditions.

Antibodies, Catalytic↗
PubMed1997

DNA- and RNA-hydrolyzing antibodies from the blood of patients with various forms of viral hepatitis.

Antibodies (Abs) hydrolyzing proteins, DNA, and RNA are detected in the blood of patients with various autoimmune diseases. In the present work, homogeneous preparations of IgG Abs from the blood of the healthy donors as well as patients with A, B, C, and delta types of viral hepatitis, influenza, pneumonia, tuberculosis, tonsillitis, duodenal ulcer, and some types of cancer were purified. For the first time, the fraction of IgG and its Fab fragments of patients with viral hepatitis were shown to have high DNA- and RNA-hydrolyzing activity. In case of Abs from the healthy donors and patients with other diseases, high activity of Abs was not detected. The data obtained by various methods indicate that the activity of hepatitis Abs is an intrinsic property of the immunoglobulins. The relative rates of hydrolysis of cCMP, poly(U), poly(A), poly(C), and tRNA(Phe) by hepatitis Abs were compared with those of RNase A and other RNases from human blood. Significant differences in activities of Abs and nucleases in hydrolysis of model substrates were demonstrated. Thus, catalytically active Abs can appear in the blood of patients not only with autoimmune disorders, but with viral diseases as well.

Antibodies, Antinuclear↗
PubMed1996

The algorithm of estimation of the Km values for primers in DNA synthesis catalyzed by human DNA polymerase alpha.

DNA synthesis with various deoxyribo homo- and heterooligoprimers in the presence of complementary templates was investigated. The lengthening of d(pN)n, primers (n = 1-10) by a unit resulted in an increase of the primer affinity and a maximal rate of polymerization. The coefficient of the affinity enhancement of primers due to formation of one hydrogen bond between primer and template was found to be 1.35. The dependence of the primer affinity and polymerization rate on template-primer structure in solution was analyzed and the objective laws of the changes of the KM and Vmax values were revealed.

Algorithms↗
PubMed1996

Interaction of human immunodeficiency virus type 1 reverse transcriptase with primer tRNALys3 and affinity modification of the enzyme by tRNALys3 derivatives.

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity. In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Kd values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.

Animals↗
PubMed1996

Recognition of the primers containing different modified nucleotide units by the Klenow fragment of DNA polymerase I from E coli.

A comparison of Km values and maximal rates of extension (Vmax) for primers containing different modified bases or mismatches, and fully complementary primers of the same length catalyzed by the Klenow fragment of E coli DNA polymerase I was carried out. Base modifications include T-T dimers and apurinic sites. In the case of mismatch, the number of complementary bases from the 3'-terminus to the non-complementary nucleotide determines the efficiency of substrate incorporation, which is a measure of degree of interaction of the enzyme with its primer template. Differently, removal of one base in any position from the 3'-terminus of the primer is equivalent to shortening of the primer by one nucleotide unit, and decreases the affinity to the enzyme by 1.8-fold. Since apurinic sites fail to interfere with the efficiency of DNA synthesis, we suppose that the Klenow fragment of E coli DNA polymerase I does not participate in the correction of DNAs containing apurinic nucleotides units. Finally, the efficiency of elongation of the d(p primer was shown to decrease with an increase in T-T dimers in the primer. When the d(pT)10m primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.

Apurinic Acid↗
PubMed1996

Phosphorylation of different human milk proteins by human catalytic secretory immunoglobulin A.

The ability of secretory immunoglobulin A (slgA) from milk of healthy mothers to phosphorylate various milk proteins in the presence of gamma-[32P]-ATP was shown to be a property of the antibodies. The polyclonal slgA was purified by sequential chromatography on Protein-A Sepharose and DEAE-cellulose, and then separated by chromatography on the ATP-Sepharose. The preparations containing all milk proteins except for protein kinases [integrated milk proteins (IMP)] were obtained by extraction of the kinase activities from the milk using their affinity to the insoluble crosslinked staphylococcus. Addition of slgA fractions (having a different affinity to ATP) to the IMP led to phosphorylation of casein and several other milk proteins with different efficiencies.

Adenosine Triphosphate↗
PubMed1995

High-affinity interaction of human immunodeficiency virus type-1 reverse transcriptase with partially complementary primers.

The comparison of Km and Vmax values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase was carried out. The primers were: (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3' end or (c) non-complementary. Non-complementary primers were not elongated by HIV-1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3' end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and Vmax, was found in the case of oligonucleotides containing non-complementary bases at the second or third position from the 3' end of the primer. The efficiency of discrimination by HIV-1 reverse transcriptase between matched and mismatched base-paired primers was about 1-1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT)4(dCdG)k(dT)4 showed higher affinity for the enzyme than (dT)4 or (dT)8 primers. These data suggest that HIV-1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5'-end region of the non-complementary primer. In addition, using tRNA(3Lys), the natural primer of HIV-1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5' end of tRNA. Thus, besides the normal binding site for the 3' end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5' end of the tRNA also interacts with a specific site on the enzyme.

DNA, Complementary↗
PubMed1995

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV-1 reverse transcriptase: tRNA(Lys,3) activation.

HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.

DNA↗
PubMed1995

High affinity interaction of mammalian DNA topoisomerase I with short single- and double-stranded oligonucleotides.

The interaction of DNA topoisomerase I (topo I) with a set of single- and double-stranded oligonucleotides containing 5-27 mononucleotides was investigated. All single- and double-stranded oligonucleotides were found to inhibit competitively the supercoiled DNA relaxation reaction catalyzed by topo I. The enzyme affinity for specific sequence pentanucleotides of the scissile (GACTT, Ki = 2 microM) and non-cleaved chain (AAGTC, Ki = 110 microM) is about 2-4 orders of magnitude higher than that for non-specific oligonucleotides. This specific sequence affinity increases in several cases; lengthening of single-stranded oligonucleotides, formation of stable duplexes between complementary oligonucleotides and preincubation of the enzyme with ligands before addition of supercoiled DNA. We assume that oligonucleotides having a high affinity to the enzyme can offer a unique opportunity for rational design of topoisomerase-targeting drugs.

Base Sequence↗
PubMed1995

Modified substrates as probes for studying uracil-DNA glycosylase.

In order to study the mechanism of action of uracil-DNA glycosylase (UDG) from human placenta, single-stranded (ss) and double-stranded (ds) oligodeoxyribonucleotides (oligos), containing deoxyuridine (dU) and a wide variety of their analogs were used. It was shown that UDG has a twofold preference for ss oligos over ds oligos and a twofold preference for intermolecular duplexes over similar hairpin-like duplexes. The replacement of dU with 1-(beta-D-2'-deoxy-threo-pentofuranosil)uracil (xU) or 1-(beta-D-3'-deoxy-threo-pentofuranosil)uracil (tU), which results in a change in sugar hydroxyl configuration, has no influence on UDG binding to such substrates, but inhibits uracil removal. A oligo containing 2'-deoxy-2'-fluorouridine (flU), with a 3'-endo conformation of modified sugar is recognized by UDG 100-200-fold less efficiently than the natural ones. F or Br atoms or a methyl group were introduced at position 5 of a dU residue in an oligo. It was shown that the nature of a substituent at this position is essential for UDG function.

Base Sequence↗