Biochemistry (Moscow) (v.73, #9)

Aconitate hydratase of mammals under oxidative stress by L. V. Matasova; T. N. Popova (957-964).
Data on the structure, functions, regulation of activity, and expression of cytosolic and mitochondrial aconitate hydratase isoenzymes of mammals are reviewed. The role of aconitate hydratase and structurally similar iron-regulatory protein in maintenance of homeostasis of cell iron is described. Information on modifications of the aconitate hydratase molecule and changes in expression under oxidative stress is generalized. The role of aconitate hydratase in the pathogenesis of some diseases is considered.
Keywords: aconitase; citrate; oxidative stress; iron; iron-regulatory proteins; regulation of activity and expression

New findings in studies of cytochromes P450 by K. N. Myasoedova (965-969).
Cytochromes P450 represent a numerous family of heme-containing enzymes belonging to the group of monooxygenases. In prokaryotes, cytochromes P450 usually perform a plastic function, whereas in eukaryotes their functions are very diverse. Mammalian cytochromes P450 are components of membranes and are involved in biosynthesis and metabolism of many physiologically active substances; moreover, these cytochromes are unique in their ability to catalyze biotransformation of xenobiotics, i.e. metabolize substances of foreign origin (drugs, toxins, environmental pollutants). The latter promotes elimination of xenobiotics, but sometimes intermediates of their metabolism are even more toxic and dangerous than the original xenobiotics per se. Some catalytic features of cytochromes P450 still need unambiguous explanation, i.e. broad substrate specificity, diversity of catalytic reactions, and unusual kinetics. Under some conditions cytochromes P450 can produce reactive oxygen species, and this is another problem attracting increasing attention. In this respect, a recent finding in mitochondria of analogs of microsomal cytochromes P450 seems especially intriguing; it was postulated that P450 can be responsible for mitochondrial dysfunction, cell apoptosis, and pathogenesis of some diseases. In this paper the present state of the art concerning these problems is considered.
Keywords: cytochrome P450; endoplasmic reticulum; mitochondria; xenobiotics; ligands; conformation; reactive oxygen species

Polymorphonuclear leukocytes (neutrophils) are the first cells that arrive at sites of infection or injury. There, besides their microorganism-targeted effector functions, activated neutrophils secrete numerous chemoattractants that recruit other leukocyte subtypes into the inflamed tissue. First, neutrophil activation leads to the upregulation of the gene expression of several classical chemokines of the CXC and CC families. Second, neutrophil granules contain preformed intracellular storage pools of chemotactically active proteins that are rapidly released upon neutrophil degranulation. The third pathway of generation of chemotactically active proteins by activated neutrophils—shedding and concomitant proteolytic processing of a membrane protein—has recently been demonstrated in our laboratory. In this review we summarize the essential features of chemoattractant production by neutrophils and their contribution to orchestrating the recruitment of leukocyte subtypes during inflammatory response.
Keywords: neutrophils; leukocytes; chemotactically active proteins; chemokines; inflammatory response

Cytosol polyphosphates (polyPs) are the main phosphate (Pi) reserve in the yeast Saccharomyces cerevisiae. In this work, the participation of cytosol polyPs and exopolyphosphatases in maintenance of Pi homeostasis under Pi deficit in the cultivation medium has been studied in different strains of S. cerevisiae. The growth of yeast strains with inactivated genes PPX1 and PPN1 encoding the yeast exopolyphosphatases and a strain with double mutations in these genes in a Pi-deficient medium is not disturbed. All the studied strains are able to maintain relatively constant Pi levels in the cytosol. In Pi-deficient medium, polyP hydrolysis in the cytosol of the parent and PPN1-deficient strains seems to be performed by exopolyphosphatase Ppx1 and proceeds without any change of the spectrum of polyP chain lengths. In the PPX1-deficient strain, long-chain polyPs are depleted first, and only then short-chain polyPs are hydrolyzed. In the double PPX1 and PPN1 mutant having low exopolyphosphatase activity, polyP hydrolysis in the cytosol starts with a notable delay, and about 20% of short-chain polyPs still remain after the polyP hydrolysis in other strains has almost been completed. This fact suggests that S. cerevisiae possesses a system, which makes it possible to compensate for inactivation of the PPX1 and PPN1 genes encoding exopolyphosphatases of the yeast cells.
Keywords: Pi depletion; inorganic polyphosphates; exopolyphosphatase; cytosol; PPX1 and PPN1 mutants; Saccharomyces cerevisiae

Effect of colchicine on sensitivity of duck salt gland Na,K-ATPase to Na+ by S. S. Yakushev; E. M. Kumskova; A. M. Rubtsov; O. D. Lopina (990-994).
Low molecular mass proteins of the FXYD family that affect the sensitivity of Na,K-ATPase to Na+ and K+ are known to be present in Na,K-ATPases in various tissues. In particular, in Na,K-ATPase from kidney a γ-subunit (with electrophoretic mobility corresponding to molecular mass of about 10 kD) is present, and Na,K-ATPase preparations from heart contain phospholemman (electrophoretic mobility of this protein corresponds to molecular mass of 13–14 kD), which provides for the interaction of heart Na,K-ATPase with cytoskeletal microtubules. Disruption of microtubules by colchicine removes phospholemman from heart Na,K-ATPase preparations. The goal of the present study was to reveal a low molecular mass protein (probably a member of FXYD family) in preparation of Na,K-ATPase from duck salt glands. Immunoprecipitation of solubilized duck salt gland Na,K-ATPase using antibodies against α1-subunit results in the coprecipitation of a 13 kD protein with the Na,K-ATPase complex. Treatment of homogenate from duck salt glands with colchicine removes this protein from the purified preparation of Na,K-ATPase. Simultaneously, we observed a decrease in the sensitivity of Na,K-ATPase to Na+ at pH 6.5. However, colchicine treatment of homogenate from rabbit kidney does not affect either the sensitivity of Na,K-ATPase obtained from this homogenate to Na+ or the content of 10 kD protein (presumably γ-subunit). The data suggest that phospholemman (or a similar member of the FXYD family) tightly interacts with Na,K-ATPase from duck salt glands and binds it to microtubules, simultaneously participating in the regulation of the sensitivity of Na,K-ATPase to Na+.
Keywords: Na,K-ATPase; colchicine; FXYD family proteins; duck salt glands

Role of the C-terminus in folding and oligomerization of bacteriophage T4 gene product 9 by L. P. Kurochkina; A. Yu. Vishnevskiy; V. V. Mesyanzhinov (995-999).
Bacteriophage T4 gene product 9 (gp9) is a structural protein of baseplate that plays a key role at the beginning of the infection process. Biologically active gp9 is a trimer that consists of three domains. It is a convenient model to study folding and oligomerization mechanisms of complex multidomain proteins. The influence of deletions and mutations of several amino acid residues in the C-terminal part of molecule on protein folding, oligomerization, and functional activity has been studied. It was determined that gp9 trimerization occurs post-translationally. It was shown that Gln282 and Ile284 are essential for gp9 trimer stabilization. The disruption of hydrogen bonds formed by Gln282 with Leu203 and Thr205 of neighboring chain has effect not only on interaction between monomers within trimer but also on folding of the polypeptide chain. Tsf (temperature sensitive for folding) and su (suppressor) mutations in the C-terminal region of the polypeptide chain affecting protein folding have been found.
Keywords: bacteriophage T4; base plate; gene product 9; protein folding; protein oligomerization; tsf mutation; su mutation

Endonuclease WEN2 with an apparent molecular mass 21.5 kD was isolated from subcellular vesicular fraction obtained from aging apoptotic coleoptiles of 8-day-old etiolated wheat seedlings and partially characterized. Similar to wheat endonuclease WEN1 of the same origin described earlier, the WEN2 enzyme is a neutral Ca2+,Mg2+,Mn2+-dependent endonuclease. Both enzymatic activities were found also in nuclei from the same cells. Mn2+ activates WEN2 more efficiently than Mg2+ or Ca2+. High ionic strength, Zn2+, and EDTA inhibit the enzyme completely. In the presence of Mg2+, elevated WEN2 activity was observed at pH between 5.5 and 7.7 and at 37°C, and without Mg2+ added it was observed in narrower pH range (from pH 6.8 to pH 7.7). The enzyme is active even at high temperature (65°C). WEN2 splits preferentially unmethylated, but WEN1 — methylated λ phage DNA. Double-stranded DNA is a preferential substrate to be hydrolyzed with WEN2. S-Adenosyl-L-methionine (SAM) significantly activates endonuclease WEN2 (the optimal SAM concentration is 0.3 mM). Contrary to strong stimulating action on WEN1, the competitive inhibitors of the DNA methylation reaction (SAM analogs S-adenosyl-L-homocysteine and S-isobutyladenosine) at concentration 0.3 mM increase WEN2 activity slightly. It is suggested that WEN2 may take part in apoptotic DNA degradation. Thus, in plants there are endonucleases that recognize methylation status of substrate DNAs and are modulated by the methyl group donor, SAM, in different fashions. Therefore, all this may indicate the presence of a restriction-modification (R-M) system in higher plants.
Keywords: apoptosis; S-adenosyl-L-methionine; aging; DNA methylation; endonuclease; mitochondria; nucleus; plant; wheat

A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5′-3′-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared. Primer elongation (polymerase reaction) is detected by changes in SYBR Green I fluorescence upon binding to dsDNA during reaction; nuclease activities are detected by changes in fluorescence due to cleavage of the probe, containing the reporter fluorophore and fluorescence quencher, and hybridized in advance to the template single-stranded region. It was also shown that the method can be used for determination of relative activities of DNA polymerase preparations, estimation of temperature-time dissociation parameters of polymerase complexes with specific antibodies to its active center, and analysis of effects of inhibitors and activators of different nature on reaction rates of dsDNA polymerization and 5′-3′-exonuclease cleavage by polymerase. The method can be also used for estimation of endonuclease activities of DNA polymerases.
Keywords: fluorescence detection; DNA polymerase activities; hot-start; nuclease activity

Sulfated galactofucan from Lobophora variegata: Anticoagulant and anti-inflammatory properties by V. P. Medeiros; K. C. S. Queiroz; M. L. Cardoso; G. R. G. Monteiro; F. W. Oliveira; S. F. Chavante; L. A. Guimaraes; H. A. O. Rocha; E. L. Leite (1018-1024).
Sulfated polysaccharides (fucans and fucoidans) from brown algae show several biological activities, including anticoagulant and anti-inflammatory activities. We have extracted a sulfated heterofucan from the brown seaweed Lobophora variegata by proteolytic digestion, followed by acetone fractionation, molecular sieving, and ion-exchange chromatography. Chemical analyses and 13C-NMR and IR spectroscopy showed that this fucoidan is composed of fucose, galactose, and sulfate at molar ratios of 1:3:2. We compared the anticoagulant activity of L. variegata fucoidan with those of a commercial sulfated polysaccharide (also named fucoidan) from Fucus vesiculosus and heparin. The experimental inflammation models utilized in this work revealed that fucoidan from L. variegata inhibits leukocyte migration to the inflammation site. Ear swelling caused by croton oil was also inhibited when sulfated polysaccharides from F. vesiculosus and L. variegata were used. The precise mechanism of different action between homo-and heterofucans is not clear; nevertheless, the polysaccharides studied here may have therapeutic potential in inflammatory disorders.
Keywords: brown algae; Lobophora variegata ; fucan; fucoidan; anti-clotting activity; anti-inflammatory activity

Co-expression of human complement regulatory proteins DAF and MCP with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects by Li Xu; Zhouzhou Zhao; Jiqun Sheng; Chengang Zhu; Hui Liu; Dahe Jiang; Xin Mao; Mingxiong Guo; Wenxin Li (1025-1030).
C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation.
Keywords: complement regulatory proteins; internal ribosome entry site (IRES); dicistronic expression vector; co-expression; hyperacute rejection

Effects of thiamine and its derivatives on inhibition of dityrosine formation were studied in reactions catalyzed by oxoferryl forms of hemoglobin. At high thiamine concentrations a complete inhibition of dityrosine formation was observed due to interaction of tyrosyl radicals with thiamine tricyclic and thiol forms. In neutral and alkaline media, tyrosyl radicals oxidized thiamine to thiochrome, oxodihydrothiochrome, and thiamine disulfide. In the absence of tyrosine, oxoferryl forms of hemoglobin manifested peroxidase activity towards thiamine and its phosphate esters by inducing their oxidation to disulfide compounds, thiochrome, oxodihydrothiochrome, and their phosphate esters, respectively, in neutral media. Thiamine and its phosphate esters were oxidized by both oxoferryl forms of hemoglobin, viz., $$ mathop cdot limits^ + Hbleft( {IV = O} ight) $$ (compound I with an additional radical on the globin) and Hb(IV=O) (compound II). Putative mechanisms of thiamine conversions under oxidative stress and the protective role of hydrophobic thiamine metabolites are discussed.
Keywords: thiamine; dityrosine; hemoglobin; oxoferryl forms; tyrosyl radicals; thiochrome; oxodihydrothiochrome

Interaction between DNA polymerase λ and RPA during translesion synthesis by Yu. S. Krasikova; E. A. Belousova; N. A. Lebedeva; P. E. Pestryakov; O. I. Lavrik (1042-1046).
Replication of damaged DNA (translesion synthesis, TLS) is realized by specialized DNA polymerases. Additional protein factors such as replication protein A (RPA) play important roles in this process. However, details of the interaction are unknown. Here we analyzed the influence of the hRPA and its mutant hABCD lacking domains responsible for protein-protein interactions on ability of DNA polymerase λ to catalyze TLS. The primer-template structures containing varying parts of extended strand (16 and 37 nt) were used as model systems imitating DNA intermediate of first stage of TLS. The 8-oxoguanine disposed in +1 position of the template strand in relation to 3′-end of primer was exploited as damage. It was shown that RPA stimulated TLS DNA synthesis catalyzed by DNA polymerase λ in its globular but not in extended conformation. Moreover, this effect is dependent on the presence of p70N and p32C domains in RPA molecule.
Keywords: DNA replication; translesion DNA synthesis; DNA polymerase λ; replication protein A

Soluble NTPase, differing in its properties from known proteins exhibiting NTPase activity, was purified from bovine brain to homogeneity. The enzyme has pH optimum at 7.5 and shows absolute dependence on bivalent cations and broad substrate specificity towards nucleoside-5′-tri-and-diphosphates, characteristics of apyrases. The NTPase follows Michaelis-Menten kinetics in the range of investigated substrate concentrations, the apparent K m values for UTP, ITP, GTP, CTP, CDP, and ATP being 86, 25, 41, 150, 500, and 260 μM, respectively. According to gel-filtration and SDS-PAGE data, the molecular mass of the enzyme is 60 kD. The NTPase is localized in the cytosol fraction and expressed in different bovine organs and tissues. Total NTPase activity of extracts of bovine organs and tissues decreases in the following order: liver > heart > skeletal muscle > lung > brain > spleen > kidney ≈ small intestine. The enzyme activity can be regulated by acetyl-CoA, α-ketoglutarate, and fructose-1,6-diphosphate acting as activators in physiological concentrations, whereas propionate exhibits an inhibitory effect.
Keywords: nucleoside triphosphatase; apyrase; purification; kinetic properties; specificity; regulation; subcellular localization; bovine brain