[NO-dependent modifications of nucleic acids].

This review is devoted to chemical transformations of nucleic acids and their components under the action of nitrogen oxide metabolites. The deamination reaction of bases is discussed in the context of possible competing transformations of its intermediates (nitrosamines, diazonium cations, diazotates, triazenes, and diazoanhydrides) and mechanisms of crosslink formation with proteins and nucleic acids. The oxidation and nitration of bases by NO2 is considered together with the possibility of radical transfer to domains from the base stacks in DNA. Reduction of redox potentials of bases as a result of stacking interactions explains the possibility of their reactions within nucleic acids with the oxidants whose redox potential is insufficient for the effective reactions with mononucleotides. Modifications of nucleic acids with peroxynitrite derivatives are discussed in the context of the effect of the DNA primary structure and the modification products formed on the reactivity of single bases. The possibility of reduction of nitro groups within modified bases to amino derivatives and their subsequent diazotation is considered. The substitution of oxoguanine for nitroguanine residues may result; the reductive diazotation can lead to undamaged guanine. The intermediate modified bases, e.g., 8-aminoguanine and 8-diazoguanine, were shown to participate in noncanonical base pairing, including the formation of more stable bonds with two bases, which is characteristic of the DNA Z-form. A higher sensitivity of RNA in comparison with DNA to NO-dependent modifications (NODMs) is predicted on the basis of the contribution of medium microheterogeneity and the known mechanisms of nitrosylation and nitration. The possible biological consequences of nucleic acids NODMs are briefly considered. It is shown that the NODMs under the action of nitrogen oxide metabolites generated by macrophages and similar cells in inflammations or infections should lead to a sharp increase in the number of mutations in the case of RNA-containing viruses. As a result, the defense mechanisms of the host organism may contribute to the appearance of new, including more dangerous, variants of infecting viruses.

[Inorganic nitric oxide metabolites participating in NO-dependent modifications of biopolymers].

Biogenous nitric(II) oxide (NO), the higher nitrogen oxides (NO2, isomeric N2O3 and N2O4, ONOO-, etc.) that are NO-derived in vivo, and the products of their transformations are active compounds capable of reactions with biopolymers and low-molecular metabolites. The products of these reactions are often considered to be various NO-dependent modifications (NODMs). The nitrated, nitrosylated, nitrosated, and other NODMs play key roles in the regulation of the most important biochemical processes. In this review, we briefly discuss the metabolic reactions of nitrogen oxides that supply active intermediates for NODMs, the NODM reaction products, and some mechanisms of NODM reparation that allow the recovery of chemically intact biopolymer molecule from a modified (chemically damaged) NODM. For example, residues of 3-nitrotyrosine arising due to the NODM reactions of proteins can be reduced to unsubstituted Tyr residues as a result of alternative NODM reactions through intermediate diazotyrosine derivatives. The heterogeneity of a medium in vivo is an important factor controlling the proceeding of NODM reactions. We showed that many processes determining NODM efficiency proceed differently in the heterogeneous media of organisms and in homogeneous aqueous solutions.

Mechanism of perfluoroalkyl halide toxicity: catalysis of perfluoroalkylation by reduced forms of cobalamin (vitamin B12).

Perfluoroalkyl halides (PFHs) are synthetic products widely used in various fields. Perfluorooctyl bromide (PFB) is used in medicine as a component of blood substitutes and for artificial lung ventilation. In both cases, it is considered a completely inert compound acting as a solvent for oxygen. However, there are many reports of PFH-induced intoxication, including lethal cases. Mechanisms underlying toxic effects of this compound remain unknown. In this study, we demonstrate that the reduced form of cobalamin (vitamin B12) typical for B12-dependent enzymes can catalyze the reactions of perfluoroalkylation, aromatic substitution, or addition by double bonds. Synthesis of perfluoro derivatives from PFHs during catalysis by cob(I)alamin-like super nucleophiles is a new possible mechanism responsible for in vivo formation of highly toxic compounds from "chemically inert" substances widely used in medicine. Catalytic perfluoroalkylation might possibly contribute to nitric oxide depletion and modulation of activity of guanylate cyclase, cytochromes, NO-synthases, and other heme-containing proteins.

[Environment of tryptophan residues in proteins--a factor for stability to oxidative nitrosylation. I. Analysis of primary structure]. / Okruzhenie ostatkov triptofana v belkakh--faktor ustoichivosti k okislitel'nomu nitrozilirovaniiu. I. Analiz pervichnykh struktur.

Micellar catalysis under aerobic conditions effectively accelerates oxidative nitrosylation because of solubilization of NO and O2 by protein membranes and hydrophobic nuclei. Nitrosylating intermediates NOx (NO2, N2O3, N2O4) form mainly in the hydrophobic phase, and therefore their solubility in aqueous phase is low and hydrolysis is rapid, local concentration of NOx in the hydrophobic phase being essentially higher than in aqueous. Tryptophan is a hydrophobic residue and can nitrosylate with the formation of isomer N-nitrosotryptophans (NOW). Without denitrosylation mechanism, the accumulation of NOW in proteins of NO-synthesizing organisms would be constant, and long-living proteins would contain essential amounts of NOW, which is however not the case. Using Protein Data Bank (more than 78,000 sequences) we investigated the distribution of tryptophan residues environment (22 residues on each side of polypeptide chain) in proteins with known primary structure. Charged and polar residues (D, H, K, N, Q, R, S) are more incident in the immediate surrounding of tryptophan (-6, -5, -2, -1, 1, 2, 4) and hydrophobic residues (A, F, I, L, V, Y) are more rare than in remote positions. Hence, an essential part of tryptophan residues is situated in hydrophilic environment, which decreases the nitrosylation velocity because of lower NOx concentration in aqueous phase and allows the denitrosylation reactions course via nitrosonium ion transfer on nucleophils of functional groups of protein and low-molecular compounds in aqueous phase.

[Biogenic nitric oxide: ten years of the second advent. Prehistory of discovery of the arginine-dependent NO biosynthesis]. / Biogennyi oksid azota: desiat' let vtorogo prishestviia. Predystoriia otkrytiia argininzavisimogo biosinteza NO.

This review has been written on the occasion of the decennial anniversary of the discovery of the arginine-oxygenase pathway for nitric oxide (NO) biosynthesis. This was one of the greatest and the most surprising discoveries in modern biochemistry and won the Nobel prize in 1998. The main steps of early studies are tracked. Many facts had pointed to the existence of biogenic NO long before 1987 but had been disregarded for reasons of psychology: it was hard to accept that NO is an assignable metabolite, since too many "inviolable" views had to be revised in this case.

Competition involving biogenic NO.

This review considers competitive reactions of NO biosynthesis and its interaction with its targets including enzyme competition for arginine, competition between the substrates in the NO-synthase active sites, competitive sources of arginine, multiple states of NO-synthases resulting from competition of reducible cofactors (FAD, FMN, BH4, heme), competition between aqueous and lipophilic phases for NO resulting in efficient micellar catalysis, and other aspects of competition involving biogenic NO.

NO catastrophes in vivo as a result of micellar catalysis.

Micellar catalysis plays a crucial role in NO metabolism because media in vivo are heterogeneous and the concentration of NO in different phases at different levels of solubility differs by degrees of magnitude. The relative volumes of the hydrophobic phases are usually small. At small volumes (which are calculated) of these phases the reaction rates of NO metabolism change. The dependence on the relative volumes is resonance-like. Not only regulation, but bifurcations and catastrophes are possible in vivo as a result of this changing due to the small change of effectiveness of micellar catalysis.

[ANSA analysis. II. Aminonaphthalenesulfonamides--detecting groups for polysubstrate analysis of proteases]. / ANSA-analiz. II. Aminonaftalinsul'fonamidy--detektiruemye gruppy dlia polisubstratnogo analiza proteaz.

The properties and synthetic methods of aminonaphthalenesulfonamides (ANSA) used as detectable groups of protease substrates are described. A list of chemical and physical properties of seventeen 5.1-ANSA with simple substituents is presented. A comparison of condition for the introduction and removal of acyl protecting groups (acetyl, trifluoroacetyl, phthaloyl, carbobenzoxy) used in ANSA synthesis is given. Examples of applicability of nitronaphthalenesulfonamides as intermediate compounds are given. The possibility of ANSA alkylation at both N(C) and N(S) is demonstrated. Substituted ANSA--sulfonylaziridenes--are used for the production of water-soluble derivatives containing the alcoxy group in the sulfonamide fragment. Criteria for the selection of detectable groups for polysubstrate analysis are discussed. Eighteen typical procedures for ANSA synthesis according to the schemes discussed are presented.

[ANSA analysis. I. Mixtures of substrates in analyzing proteases: features of the kinetics of hydrolysis during competition]. / ANSA-analiz. I. Smesi substratov v analize proteaz: obsobennosti kinetiki gidroliza pri nalichii konkurentsii.

Studies designed to investigate the utility of mixtures of chromogenic peptide substrates for protease analysis are reviewed. Individual applications of aminonaphthalenesulfonamide substrates with a variable structure of the leaving group are discussed. The kinetic curves describing the mixtures of competing substrates are presented and the conditions of the occurrence of S-shaped curves specified. The kinetic differences for mixtures of competing substrates split by a single enzyme and for those independently split by various substrates are demonstrated. A comparison between experimental and calculated kinetic curves is made. The cause of erratic results obtained in a number of studies using substrate mixtures is indicated.

[ANSA analysis. III. Synthesis of aminonaphthalinesulfonamide chromogenic substrates for protease analysis]. / ANSA-analiz. III. Sintez aminonaftalinsul'fonamidnykh khromogreenykh substratov dlia analiza proteaz.

A review of synthetic methods of peptide substrates containing aminonaphthalenesulphonamide (ANSA) as the detected leaving group is presented. Variations of aminoacylic and peptide ANSA derivatives using ANSA as the C-protect group at all stages of the peptide synthesis, condensations of the ANSA with the N-protected peptide fragment obtained preliminary, the application of aminoacyl-ANSA as syntones are discussed. The synthesis scheme used while determining optimal ANSA substrates that involves reactions of aminoacyl derivatives of aminonaphthalenesulfonylchlorides with amines is shown. The application of di-tert-butylpyrocarbonate, DCC, chlorodimethylformiminium chloride, alkylchloroformate as condensing agents is described. The protection of amino groups was carried out by using Boc- and Cbz- groups.

[ANSA analysis. IV. Specificity spectra in characterizing proteases and mixtures of them]. / ANSA-analiz. IV. Spektry spetsifichnosti v kharakteristike proteaz i ikh smesei.

ANSA-analysis was used for characterization of proteases and their mixtures, such as snake venoms. The method is based on the cleavage by proteases of mixtures of competing chromogenic substrates containing substituted aminonaphtalenesulfonamide (ANSA) detectable groups. All detectable ANSA groups in the substrate mixtures have non-identical modifiers, one or two substituents in the sulfonamide fragment and can be determined by chromatographic methods. To identify venoms, a mixture of six peptide substrates cleaved at the Arg-ANSA bond was proposed. Hydrolysis of this substrate mixture catalyzed by the venoms of different Crotalidae and Viperidae species gave characteristic chromatograms (ANSA spectra) for each tested sample. A method for quantitative description of differences in ANSA spectra has been proposed. Each ANSA spectrum can be presented as a vector going from the origin of the coordinated axes to a point in an n-dimensional space (n is the number of assayed ANSA products of proteolysis) with peak squares of corresponding ANSA as coordinates. The similarity between two ANSA spectra will then be characterized by angle between their vectors.

[ANSA analysis. V. Secondary specificity of peptidases to the structure of detected groups of aminonaphthalenesulfamide chromogenic substrates]. / ANSA-analiz. V. Vtorichnaia spetsifichnost' peptidaz k strukture detektiruemykh grupp aminonaftalinsul'famidnykh khromogennykh substratov.

Mixtures of competing aminonaphthalenesulfonamide (ANSA) substrates can be used for identifying secondary substrate specificity of peptidases and their mixtures. All substrates in the mixture had identical amino acid residues; however, cleaved ANSA differed in chromatographic mobilities due to differences in the substituent in the sulfonamide residue. ANSA generated by limited proteolysis of the substrate mixture by the enzyme preparation were detected chromatographically. The chromatogram obtained (the ANSA spectrum) was characteristic of each enzyme or enzyme-containing preparation. Using arginyl-ANSA mixtures, informative ANSA spectra suitable for enzyme identification were obtained. The correlations between the structure of the substituents in the SONR1R2 group of ANSA and the efficiency of substrate hydrolysis were studied. The method was used to identify snake venoms.

[ANSA analysis. VI. Activation, inhibition and interaction of proteases in enzyme mixtures]. / ANSA-analiz. VI. Aktivatsiia, ingibirovanie i vzaimodeistviia proteaz v smesiakh fermentov.

A new accurate method (ANSA-analysis) is used for studying the interactions of proteases with their inhibitors or other proteases. The method is based on the cleavage by proteases of mixtures of competing chromogenic substrates containing aminonaphthalenesulfonamide (ANSA) detectable groups. Each substrate contained a specifically substituted ANSA group which showed its specific retention time during chromatographic separation. For the analysis of blood coagulation, mixtures of blood-clotting factor substrates were used. Hydrolysis of the substrate mixture catalyzed by blood samples gave characteristic chromatograms (ANSA spectra) for each sample. The activation time before injection of the blood sample into the substrate mixture and the pool of clotting factors and inhibitors both had influence upon the ANSA spectrum. The ANSA spectra of mixtures of trypsin and/or chymotrypsin with snake venoms are described as A x (the ANSA spectrum of a protease) + B x (the ANSA spectrum of a venom) + C x (the ANSA spectrum of catalytically active interaction products). They are additive (A = B = 1, C = 0), if no proteolysis, inhibition or activation takes place. ANSA spectra analysis shows deviations from additivity for some mixtures of Viperidae, (including E. carinatus), Naja naja, Agkistrodon contortrix and A. halys venoms. Explanations for the inability to detect inhibitors in venoms having a high protease activity by previously used methods are given.

Age changes of Vipera berus venom amidolytic activity.

The study of age changes of amidolytic activity of Vipera berus venom is investigated using mixtures of chromogenic peptide substrates differing by detected groups. Quantities of venom (total protein content) and its proteolytic activity from snakes of different ages were compared. The venom composition of newly born adders was shown to be considerably different from the venom composition of young (12-month) adders of the same population. The next evolution of protease activity is weakly expressed.

[A method of screening artificial substrates for proteolytic enzymes]. / Sposob skrininga iskusstvennykh substratov proteoliticheskikh fermentov.

A method of screening of proteolytic enzyme's substrates is proposed. An equimolar mixture of substrates consisting of peptide and easily detectable chromophore moieties (all chromophores in the mixture must be different) is subjected to enzymatic treatment. The cleaved chromophore groups, which are products of the substrate proteolysis, are quantitatively determined by chromatography. The Kcat/Km ratio is greater for substrates with higher initial rate accumulation of proteolysis products. The method is illustrated by screening of peptide derivatives of aminonaphtalene sulphonamides for trypsin assay. Proteolysis products are determined by HPLC with absorption detection or by TLC with fluorescence detection.

[A possible approach to the analysis of protease mixtures]. / Vozmozhnyi podkhod k analizu smesei proteaz.

A problem of quantitative assay of proteases in complex mixtures is solved by using a set of peptide substrates with detectable (chromogenic or fluorogenic) groups (DGs). Quantitation of separate DGs released in reaction of enzyme mixture with a set of substrates is carried out in chromatographic analysis of reaction products. Reaction of peptide derivatives of aminonaphthalene sulfamides with a mixture of thrombin and proteases from viper venom shows the amounts of produced DGs to be proportional to the amounts of both thrombin and venom proteases, confirming the validity of proposed approach. There are cases of mutual influence of some components in proteases mixtures as illustrated by inhibition of trypsin activity in presence of viper venom; one determines enzyme activities in this specific mixture rather than their amounts.

DNA-dependent RNA polymerase from the chlorotetracycline producing strain of Streptomyces aureofaciens.

RNA polymerase from Streptomyces aureofaciens has been purified by polyethyleneimine precipitation followed by chromatography first on DEAE-cellulose, then heparin-Sepharose and finally on an aminooxybutylcellulose matrix containing immobilised S. aureofaciens DNA. The enzyme is composed of three subunits of approximately 145, 136 and 44 kDa that are in a ratio of approx. 1:1:2. In many isolations two additional subunits of approximately 68 and 39 kDa and some minor protein bands of approximately 110, 85 and 61 kDa are also present. Thus, the structure of this enzyme is very similar to other bacterial RNA polymerases, exhibiting an alpha 2 beta beta' core and the additional proteins rho and sigma.

Purification and characterization of tRNA (adenine-1-)-methyltransferase from Thermus flavus strain 71.

tRNA (adenine-1-)-methyltransferase was isolated from the extreme thermophile Thermus flavus, strain 71. It was purified about 2000-fold by ammonium sulfate fractionation and affinity chromatography on tRNA bound to aminohydroxybutylcellulose via its oxidized 3' end. The purified protein preparation is free of nuclease and aminoacyl-tRNA synthetase activity and contains no more than 4% of tRNA (guanine-7-)methyltransferase activity. The only activity of the enzyme is to methylate A58 in the T psi loop of tRNA. Out of the eight purified tRNAs examined, only yeast tRNATrp was not utilized as a substrate. The enzyme is highly thermostable. It is most active at 75 degrees C. tRNA (adenine-1-)-methyltransferase has a Km of 0.4-0.5 microM for tRNA2Gln from Escherichia coli and a Km of 6 microM for S-adenosyl-L-methionine.

[Amino oxyadsorbents. New type of adsorbents for affinity chromatography: purification of tRNA-methylases from rat nephron on aminooxybutylcellulose with immobilized tRNA]. / Aminooksiadsorbenty. 8. Novyi tip sorbentov dlia affinnoi khromatografii: ochistka tRNK-metilaz nefromy krysy na aminooksibytiltselliuloze s immobilizovannoi tRNK.

A new type of sorbents for affinity chromatography is suggested and used to purify tRNA methylases. tRNA was immobilized on aminooxybutylcellulose via the oxidized 3'-end. In order to bind other enzymes specific for nucleic acids in general, e. g. nucleases, and to achieve a higher degree of purification the crude enzyme preparation was treated with rRNA immobilized on aminooxybutycellulose. The sequential application of two sorbents mentioned allows to get an approximately two hundred fold purification of total tRNA methylases. In a separate experiment the possibility of individual tRNA methylase fractionation by means of elution with a NACl gradient was shown; the degree of purification for some methylases was more than a thousand fold.