Biochemistry (Moscow) (v.75, #5)

Lysine methylation of nonhistone proteins is a way to regulate their stability and function by K. S. Egorova; O. M. Olenkina; L. V. Olenina (535-548).
This review is devoted to the dramatically expanding investigations of lysine methylation on nonhistone proteins and its functional importance. Posttranslational covalent modifications of proteins provide living organisms with ability to rapidly change protein activity and function in response to various stimuli. Enzymatic protein methylation at different lysine residues was evaluated in histones as a part of the “histone code”. Histone methyltransferases methylate not only histones, but also many nuclear and cytoplasmic proteins. Recent data show that the regulatory role of lysine methylation on proteins is not restricted to the “histone code”. This modification modulates activation, stabilization, and degradation of nonhistone proteins, thus influencing numerous cell processes. In this review we particularly focused on methylation of transcription factors and other nuclear nonhistone proteins. The methylated lysine residues serve as markers attracting nuclear “reader” proteins that possess different chromatin-modifying activities.
Keywords: lysine; methylation of nonhistone proteins; p53; posttranslational modification; Stellate

Synthetic water-soluble phenolic antioxidant regulates L-arginine metabolism in macrophages: A possible role of Nrf2/ARE by V. O. Tkachev; E. B. Menshchikova; N. K. Zenkov; N. V. Kandalintseva; N. N. Volsky (549-553).
Synthetic water-soluble phenolic antioxidant TS-13 exhibits pronounced anti-inflammatory properties in vivo and induces intracellular signal system Nrf2/ARE. At concentrations 150–1000 μM it inhibits nitric oxide (NO) production in mouse peritoneal macrophages. However, this compound at low concentrations (1–100 μM) paradoxically increases NO production and decreases activity of arginase. These results are indicative of an ambiguous role of NO and its metabolites in the mechanism of development of inflammatory reaction.
Keywords: nitric oxide; arginase; macrophages; inflammation; antioxidant responsive element; Nrf2; TS-13

Binding of sterols affects membrane functioning and sphingolipid composition in wheat roots by Yu. N. Valitova; E. R. Kotlova; A. V. Novikov; A. L. Shavarda; K. A. Artemenko; R. A. Zubarev; F. V. Minibayeva (554-561).
The present work was devoted to the exploration of the role of sterols in the functioning of membranes in root cells. Membrane characteristics and composition of the membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with exogenous cholesterol and antibiotic nystatin, which specifically binds with endogenous sterols, were analyzed. Cholesterol caused a fall of membrane potential, acidification of the incubation medium, decrease in potassium leakage of roots, and increase in the level of exogenous superoxide radical. Similarly to cholesterol, the application of nystatin also induced the depolarization of the plasma membrane, but in contrast with cholesterol it was accompanied by alkalinization of the incubation medium and decrease in the level of exogenous superoxide radical. Analysis of membrane lipids showed that following nystatin treatment the total sterol content in roots did not change, while the level of complex sphingolipids represented mainly by glycoceramides became higher. Using mass spectrometry with electrospray ionization (+ESI-MS) for the analysis of the glycoceramide composition, we showed that nystatin induced changes in the ratios of molecular species of glycoceramides. It was suggested that the modification of the sterol component of plasma membrane could influence membrane functioning by changing the sphingolipid composition of lipid rafts.
Keywords: wheat roots; nystatin; sterols; glycoceramides; reactive oxygen species; mass spectrometry with electrospray ionization

Enzymes of naphthalene metabolism by Pseudomonas fluorescens 26K Strain by N. A. Leneva; M. P. Kolomytseva; B. P. Baskunov; L. A. Golovleva (562-569).
The ability of Pseudomonas fluorescens 26K strain to utilize naphthalene at concentrations up to 600 mg/liter as the sole source of carbon and energy in mineral liquid media was shown. Using HPLC, TLC, and mass-spectrometry, the intermediates of naphthalene transformation by this strain were identified as naphthalene cis-1,2-dihydrodiol, salicylaldehyde, salicylate, catechol, 2-hydroxymuconic semialdehyde, and 1-naphthol. Catechol 2,3-dioxygenase (a homotetramer with native molecular mass 125 kDa) and NAD+-dependent homohexameric naphthalene cis-1,2-dihydrodiol dehydrogenase with native molecular mass 160 kDa were purified from crude extract of the strain and characterized. NAD+-dependent homodimeric salicylaldehyde dehydrogenase with molecular mass 110 kDa was purified and characterized for the first time. Based on the data, a pathway of naphthalene degradation by P. fluorescens 26K is suggested.
Keywords: polyaromatic hydrocarbons; naphthalene; biodegradation; catechol 2,3-dioxygense; naphthalene cis-1,2-dihydrodiol dehydrogenase; salicylaldehyde dehydrogenase

In the present work the role of conserved residue E429 of cytochrome P45011A1 has been studied. The charge neutralization of E429Q results in 3-fold decrease of K d as well as V max compared to the wild type hemoprotein indicating tighter binding and, as the result, the impaired dissociation of oxidized adrenodoxin from the complex. As cytochrome P45011A1-adrenodoxin complex formation is driven primarily by electrostatic interactions, the low activity of E429Q mutant is completely restored to that of wild type hemoprotein by increasing of ionic strength. The charge neutralization of the corresponding residue of rat cytochrome P45011B2 has the same effect: the activity is 10-fold decreased but it is restored by increasing of ionic strength without effect on the ratio of products formed. Thus, this is the first report on identification of residues involved in modulation of dissociation of redox partner from the complex with cytochrome P450s.
Keywords: cytochrome P450; cytochrome P45011A1; cytochrome P45011B2; adrenodoxin; electron transfer; site-directed mutagenesis; protein-protein interactions

Electron transfer between exogenous electron donors and reaction center of photosystem 2 by M. D. Mamedov; V. N. Kurashov; I. O. Petrova; A. A. Zaspa; A. Yu. Semenov (579-584).
Transfer of electrons between artificial electron donors diphenylcarbazide (DPC) and hydroxylamine (NH2OH) and reaction center of manganese-depleted photosystem 2 (PS2) complexes was studied using the direct electrometrical method. For the first time it was shown that reduction of redox-active amino acid tyrosine Y z · by DPC is coupled with generation of transmembrane electric potential difference (δΨ). The amplitude of this phase comprised ∼17% of that of the δΨ phase due to electron transfer between YZ and the primary quinone acceptor QA. This phase is associated with vectorial intraprotein electron transfer between the DPC binding site on the protein-water interface and the tyrosine Y z · . The slowing of ΔΨ decay in the presence of NH2OH indicates effective electron transfer between the artificial electron donor and reaction center of PS2. It is suggested that NH2OH is able to diffuse through channels with diameter of 2.0–3.0 Å visible in PS2 structure and leading from the protein-water interface to the Mn4Ca cluster binding site with the concomitant electron donation to Y z · . Because the dielectrically-weighted distance between the NH2OH binding site and Y z · is not determined, the transfer of electrons from NH2OH to Y z · could be either electrically silent or contribute negligibly to the observed electrogenicity in comparison with hydrophobic donors.
Keywords: reaction center; photosystem 2; proteoliposomes; photopotential; diphenylcarbazide; hydroxylamine; channels

Protein-lipid particles of medicinal leech salivary gland secretion; Their size and morphology by I. P. Baskova; T. G. Yudina; L. L. Zavalova; A. S. Dudkina (585-589).
The relative location of proteins and lipids in particles of medicinal leech salivary gland secretion (SGS) is revealed for the first time. Their sizes and morphology are described. Using scanning electron microscopy and transmission electron microscopy, it was determined that SGS consists of particles of different sizes and form. This picture is supported by confocal laser scanning microscopy of SGS preparations treated with fluorescein isothiocyanate. After incubation with nonionic detergents (Brij 35 and Tween 20), transmission electron microscopy revealed the dissociation of fragments composing protein-lipid particles (PLP), and in this case an increase in free protein concentration determined by a modification of the Lowry method was observed. Perylene probing of lipids in SGS preparations showed that they are concentrated mainly inside PLP and are almost absent on the surface. Cholesterol was detected during SGS probing using the cholesteryl-Bodipy (hydrophobic fluorescent analog of cholesterol) on surface sections during confocal analysis of electron microphotographs of SGS. This analysis detected PLP structures in SGS resembling caveoles full of cholesterol. SGS, preliminary frozen at −70°C, transformed into a multitude of similar small particles visualized by transmission electron microscopy, whose fixed distribution resembled water crystal structure.
Keywords: medicinal leech salivary gland secretion; scanning electron microscopy; transmission electron microscopy; confocal laser scanning microscopy; nonionic detergents; lipids; cholesterol

Proteomic analysis of salicylate-induced proteins of pea (Pisum sativum L.) leaves by I. A. Tarchevsky; V. G. Yakovleva; A. M. Egorova (590-597).
The effect of 50 μM salicylic acid on soluble proteins of pea (Pisum sativum L.) leaves was studied by proteomic analysis. Thirty-two salicylate-induced proteins were found, and 13 of these were identified using MALDI TOF MS. Salicylate-induced increased content was shown for the first time for the family 18 glycoside hydrolase, α-amylase, 33 kDa protein of photosystem II, lipid-desaturase-like protein, and glutamine amidotransferase. Increased content of protective proteins of direct antipathogenic action such as chitinase and β-1,3-glucanases was also noted.
Keywords: salicylic acid; proteomic investigations; salicylate-induced proteins

The effect of acetoacetate on palmitate-induced uncoupling with the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter has been studied in liver mitochondria. The incubation of mitochondria with acetoacetate during succinate oxidation in the presence of rotenone, oligomycin, and EGTA suppresses the accumulation of conjugated dienes. This is considered as a display of antioxidant effect of acetoacetate. Under these conditions, acetoacetate does not influence the respiration of mitochondria in the absence or presence of palmitate but eliminates the ability of carboxyatractylate or aspartate separately to suppress the uncoupling effect of this fatty acid. The action of acetoacetate is eliminated by β-hydroxybutyrate or thiourea, but not by the antioxidant Trolox. In the absence of acetoacetate, the palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter); in its presence, it is limited by a stage insensitive to the effect of these agents. In the presence of Trolox, ADP suppresses the uncoupling action of palmitate to the same degree as carboxyatractylate. Under these conditions, acetoacetate eliminates the recoupling effects of ADP and aspartate, including their joint action. This effect of acetoacetate is eliminated by β-hydroxybutyrate or thiourea. It is supposed that the stimulating effect of acetoacetate is caused both by increase in the rate of transfer of fatty acid anion from the inner monolayer of the membrane to the outer one, which involves the ADP/ATP antiporter and aspartate/glutamate antiporter, and by elimination of the ability of ADP to inhibit this transport. Under conditions of excessive production of reactive oxygen species in mitochondria at a high membrane potential and in the presence of small amounts of fatty acids, such effect of acetoacetate can be considered as one of the mechanisms of antioxidant protection.
Keywords: liver mitochondria; acetoacetate; fatty acids; uncoupling; ADP/ATP antiporter; aspartate/glutamate antiporter

Capsular polysaccharide of the bacterium Azospirillum lipoferum Sp59b: Structure and antigenic specificity by O. N. Smol’kina; V. V. Kachala; Yu. P. Fedonenko; G. L. Burygin; E. L. Zdorovenko; L. Yu. Matora; S. A. Konnova; V. V. Ignatov (606-613).
Antigenic differences were revealed between the cell wall outer membrane lipopolysaccharides and the capsular high molecular weight bioglycans for a typical strain of the nitrogen-fixing rhizobacterium Azospirillum lipoferum Sp59b using antibodies prepared against the homologous lipopolysaccharide and lipopolysaccharide-protein complex. From the capsular lipopolysaccharide-protein and polysaccharide-lipid complexes of A. lipoferum Sp59b, polysaccharides were isolated and their structure was for the first time established in Azospirillum by monosaccharide analysis which included determination of the absolute configurations, methylation, O-deacetylation, and one- and two-dimensional NMR spectroscopy. The polysaccharides of the capsular complexes were shown to have identical structure of the branched tetrasaccharide repeating unit, which differs from the structure of the O-specific polysaccharide within the outer membrane lipopolysaccharide of this strain.
Keywords: Azospirillum ; capsular antigens; polysaccharide structure

Effect of Ca2+ on programmed death of guard and epidermal cells of pea leaves by D. B. Kiselevsky; Yu. E. Kuznetsova; L. A. Vasil’ev; N. V. Lobysheva; R. A. Zinovkin; A. V. Nesov; A. A. Shestak; V. D. Samuilov (614-622).
The effect of Ca2+ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca2+ at concentrations of 1–100 μM increased and at a concentration of 1 mM prevented the CN—induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca2+ at concentrations of 0.1–1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN was suppressed by 2 mM Ca2+ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 μM concentration prevented GC death induced by CN or CN + 0.1 mM Ca2+ but had no influence on respiration and photosynthetic O2 evolution in pea leaf slices. The generation of reactive oxygen species determined from 2′,7′-dichlorofluorescein fluorescence was promoted by Ca2+ in epidermal peels from pea leaves.
Keywords: reactive oxygen species; calcium; programmed cell death; necrosis; guard cells; epidermal cells; leaves; pea

Structure of an acidic O-specific polysaccharide of the marine bacterium Pseudoalteromonas agarivorans KMM 232 (R-form) by N. A. Komandrova; V. V. Isakov; S. V. Tomshich; L. A. Romanenko; A. V. Perepelov; A. S. Shashkov (623-628).
An acidic O-specific polysaccharide containing L-rhamnose, 2-acetamido-2-deoxy-D-galactose, 2,6-dideoxy-2-(N-acetyl-L-threonine)amino-D-galactose, and 2-acetamido-2-deoxy-D-mannuronic acid was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Pseudoalteromonas agarivorans KMM 232 (R-form) followed by gel-permeation chromatography. The polysaccharide was subjected to Smith degradation to give a modified polysaccharide with trisaccharide repeating unit containing L-threonine. The initial and modified polysaccharides were studied by sugar analysis and 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, ROESY, and HSQC experiments, and the structure of the branched tetrasaccharide repeating unit of the polysaccharide was established.
Keywords: Pseudoalteromonas agarivorans ; marine bacteria; bacterial polysaccharide structure; O-specific polysaccharide; NMR spectroscopy; threonine

The goal of this work was to investigate the possible involvement of protein transcription factor Yap1 in regulation of activity of antioxidant enzymes superoxide dismutase and catalase during yeast response to nitrosative stress. It was found that the inactivation of the YAP1 gene, encoding Yap1p, cancelled the activation of superoxide dismutase and catalase by NO-donors. Then, using chimeric protein Yap1-GFP, we found the accumulation of Yap1p in the nucleus in response to nitrosative stress. Therefore, we conclude that these results in combination with previous data clearly demonstrate the involvement of Yap1p in upregulation of superoxide dismutase and catalase in yeast cells in response to nitrosative stress.
Keywords: yeast; Saccharomyces cerevisiae ; nitrosative stress; Yap1p; regulatory protein; fluorescence

Accurate mass tag retention time database for urine proteome analysis by chromatography-mass spectrometry by I. A. Agron; D. M. Avtonomov; A. S. Kononikhin; I. A. Popov; S. A. Moshkovskii; E. N. Nikolaev (636-641).
Information about peptides and proteins in urine can be used to search for biomarkers of early stages of various diseases. The main technology currently used for identification of peptides and proteins is tandem mass spectrometry, in which peptides are identified by mass spectra of their fragmentation products. However, the presence of the fragmentation stage decreases sensitivity of analysis and increases its duration. We have developed a method for identification of human urinary proteins and peptides. This method based on the accurate mass and time tag (AMT) method does not use tandem mass spectrometry. The database of AMT tags containing more than 1381 AMT tags of peptides has been constructed. The software for database filling with AMT tags, normalizing the chromatograms, database application for identification of proteins and peptides, and their quantitative estimation has been developed. The new procedures for peptide identification by tandem mass spectra and the AMT tag database are proposed. The paper also lists novel proteins that have been identified in human urine for the first time.
Keywords: proteomics; ion cyclotron resonance mass spectrometry; accurate mass time tags; urine

Biophysical characterization of a recombinant leucyl aminopeptidase from Bacillus kaustophilus by Meng-Chun Chi; Hui-Ping Chang; Gu-Gang Chang; Tzu-Fan Wang; Hsien-Bin Huang; Long-Liu Lin (642-647).
The biophysical properties of Bacillus kaustophilus leucyl aminopeptidase (BkLAP) were examined in terms of analytical ultracentrifugation, fluorescence spectroscopy, and circular dichroism. By using the analytical ultracentrifuge, we demonstrated that tetrameric BkLAP exists as the major form in solution at protein concentration of 1.5 mg/ml at pH 8.0. The native enzyme started to unfold beyond ∼1 M GdnHCl and reached an unfolded intermediate with [GdnHCl]1/2 at 1.8 M. Thermal unfolding of BkLAP was found to be highly irreversible and led to a marked formation of aggregates.
Keywords: Bacillus kaustophilus ; leucyl aminopeptidase; analytical ultracentrifuge; thermal unfolding; chemical denaturation

The effect of Ficoll 70 on the thermal stability and structure of creatine kinase (CK) was studied using far-UV CD spectra and intrinsic fluorescence spectra. The thermal transition curves monitored by CD spectra were fitted to a two-state model using a modified form of the van’t Hoff equation to obtain the transition temperature (T m) and enthalpy change (ΔH u) of thermally induced denaturation of CK in the absence and presence of Ficoll 70. An increase in T m with constant ΔH u was observed with increasing Ficoll 70 concentration, suggesting that Ficoll 70 enhances the thermal stability of CK. Fluorescence spectral measurements confirmed this protective effect of Ficoll 70 on CK structure. In addition, we observed a crowding-induced compaction effect on the structure of both native state and thermally denatured state of CK in the presence of Ficoll 70, which is more obvious on the structure of the denatured ensemble compared to that of the native ensemble. Our observations qualitatively accord with the predictions of previously proposed crowding theory for the effect of intermolecular excluded volume on protein stability and structure. These findings imply that the effects of macromolecular crowding are essential to our understanding of protein folding and unfolding occurring in vivo.
Keywords: macromolecular crowding; thermal stability; Ficoll 70; creatine kinase

Characterization of a monomeric heat-labile classical alkaline phosphatase from Anabaena sp. PCC7120 by Ming Luo; Yong-Chao Guo; Jiao-Yu Deng; Hong-Ping Wei; Zhi-Ping Zhang; Yan Leng; Dong Men; Li-Rong Song; Xian-En Zhang; Ya-Feng Zhou (655-664).
Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (Pi) during Pi starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during Pi starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase APA, coded by all2843, is characterized as a metalloenzyme containing Mg2+ and Zn2+ with molar ratio of 1: 2. Site-directed mutagenesis analysis indicated that, though the active center of APA is highly conserved in comparison with EAP, differences do exist between APA and EAP in metal ion coordination. Besides, biochemical analysis revealed that APA is a monomeric protein and inactivated rapidly at 50°C. These results suggest that APA is the first monomeric heat-labile classical PhoA found in cyanobacteria.
Keywords: Anabaena sp. PCC7120; phosphorous starvation; alkaline phosphatase; metalloenzyme; site-directed mutagenesis

Bioactive Peptides by G. Ya. Wiederschain (671-671).