Biochemistry (Moscow) (v.74, #6)

Molecular mechanisms of homocysteine toxicity by A. A. Boldyrev (589-598).
Hyperhomocysteinemia is a risk factor for a number of cardiovascular and neurodegenerative processes as well as a complicating factor in normal pregnancy. Toxic effects of homocysteine and the product of its spontaneous oxidation, homocysteic acid, are based on their ability to activate NMDA receptors, increasing intracellular levels of ionized calcium and reactive oxygen species. Even a short-term exposure of cells to homocysteic acid at concentrations characteristic of hyperhomocysteinemia induces their apoptotic transformation. The discovery of NMDA receptors both in neuronal tissue and in several other tissues and organs (including immunocompetent cells) makes them a target for toxic action of homocysteine. The neuropeptide carnosine was found to protect the organism from homocysteine toxicity. Treatment of pregnant rats with carnosine under conditions of alimentary hyperhomocysteinemia increases viability and functional activity of their progeny.
Keywords: homocysteine; homocysteic acid; NMDA receptors; neurons; lymphocytes; neutrophils

After solubilization of frog rod outer segments (ROS) with mild detergents (digitonin, n-dodecyl-β-D-maltoside, Chaps, Triton X-100) and subsequent one-dimensional blue native polyacrylamide gel electrophoresis (1D BN-PAGE), the position of rhodopsin (Rh) on the gradient gel does not match the monomer with molecular weight of 40 kDa but appears self-associated into aggregate of Rh (RhA) with molecular mass varying in different detergents from 85 to 125 kDa. Short-term treatment (∼2 h) of the excised BN-PAGE strip containing RhA by denaturing detergent mixture (10% SDS + 1 mM dithiothreitol (DTT)) followed by 2D SDS-PAGE revealed dissociation of the RhA into opsin monomer and unidentified proteins. Long-term treatment (∼2 days) of RhA that included extraction, denaturation, concentration, and electrophoresis induced, along with dissociation of RhA into opsin monomer + unidentified proteins, also formation of opsin dimers, trimers, and higher oligomers owing to a secondary aggregation of opsin. Direct solubilization of the ROS by harsh SDS + DTT detergent mixture followed by 1D SDS-PAGE revealed only opsin monomer that upon heating disappeared, trans-forming into higher oligomers owing to secondary aggregation. The data show that degree of Rh oligomerization depends on specific conditions in which it stays. In the native state in the photoreceptor membrane as well as in mild detergents frog Rh exists mainly as dimers or higher oligomers. After solubilization with denaturing detergents, RhA can dissociate into monomers that then spontaneously self-associate into higher oligomers under the influence of various factors (for example, heating).
Keywords: frog rhodopsin; oligomerization; dissociation; BN-PAGE; SDS-PAGE

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH by O. A. Antonova; S. A. Loktionova; Yu. A. Romanov; O. N. Shustova; M. V. Khachikian; A. V. Mazurov (605-612).
Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O2, 5% CO2, 93% N2) for 24 h followed by reoxygenation (21% O2, 5% CO2, 74% N2) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by ∼30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.
Keywords: endothelium; hypoxia; reoxygenation; apoptosis; cell adhesion molecules; von Willebrand factor; extracellular pH

DNA (cytosine-5-carbon) methylation is one of the hallmarks of mammalian chromatin modifications. Distinct methylation pattern can generate synergistic or antagonistic interaction affinities for CpG-islands associated with methylated or unmethylated cytosine binding proteins, which also may dictate histone modifications and dynamic transition between transcriptionally silent or transcriptionally active chromatin states. The enzymes and cofactors associated with DNA-methylation reactions are convincing in terms of chemistry and chemical thermodynamics. The mechanism of demethylation, the candidate enzyme(s) exhibiting direct demethylase activity, and associated cofactors are not firmly established. Use of azanucleosides, such as 5-azacytidine and 5-aza-2′-deoxycytidine (AzadC), in cell culture produces re-expression of certain genes, which otherwise were repressed in association with hypermethylated CpG-rich promoters. Hence the notion developed that AzadC is a demethylating agent. Here we discuss the broad global pictures with the following points: first, chemical definition and recent advances regarding the mechanism of DNA (cytosine-5-carbon) methylation (MeCpG-DNA or MeCpNpG-DNA formation) and MeCpG/MeCpNpG-DNA-demethylation, and then with the mechanistic basis of inactivation of DNA-methyltransferase 1 by AzadC. This will clarify that: (i) AzadC has nothing to do with DNA-demethylation; (ii) it cannot prevent even de novo methylation in non-replicating cells; (iii) it can only prevent replication coupled maintenance as well as de novo methylations. Finally, we would like to suggest that terming/designating AzadC as DNA-demethylating agent is a serious misuse of chemistry and chemical terminology.
Keywords: epigenetics; DNA-methylation; DNA-demethylation; DNA-methyltransferase; 5-aza-2′-deoxycytidine

High salt stress in coupled and uncoupled thylakoid membranes: A comparative study by P. Mehta; A. Jajoo; S. Mathur; S. I. Allakhverdiev; S. Bharti (620-624).
The effect of high salt concentration on photosystem II (PS II) electron transport rates and chlorophyll a fluorescence induction kinetics was investigated in coupled and uncoupled spinach thylakoid membranes. With increase in salt concentration, the rates of electron transport mediated by PS II and the F v/F m ratio were affected more in uncoupled thylakoids as compared to coupled thylakoid membranes. The uncoupled thylakoid membranes seemed to behave like coupled thylakoid membranes at high NaCl concentration (∼1 M). On increasing the salt concentration, the uncoupler was found to be less effective and Na+ probably worked as a coupling enhancer or uncoupling suppressor. We suggest that positive charge of Na+ mimics the function of positive charge of H+ in the thylakoid lumen in causing coupled state. The function of NaCl (monovalent cation) could be carried out by even lower concentration of Ca2+ (divalent cation) or Al3+ (trivalent cation). We conclude that this function of NaCl as coupling enhancer is not specific, and in general a positive charge is required for causing coupling in uncoupled thylakoid membranes.
Keywords: high salt stress; uncoupler; PS II and PS I; electron transport; thylakoid membranes

Site-directed mutagenesis of cytochrome c: Reactions with respiratory chain components and superoxide radical by T. Yu. Pepelina; R. V. Chertkova; T. V. Ostroverkhova; D. A. Dolgikh; M. P. Kirpichnikov; V. G. Grivennikova; A. D. Vinogradov (625-632).
Three forms of horse heart cytochrome c with specific substitutions of heme cleft surface located amino acid residues involved in specific interactions with ubiquinol:cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) were constructed, and their reactions with superoxide radical produced by NADH:ubiquinone reductase (complex I) were studied. The proteins with six (K27E/E69K/K72E/K86E/K87E/E90K and K8E/E62K/E69K/K72E/K86E/K87E) and eight (K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K) substitutions were inactive in the cytochrome c oxidase reaction, and their reduction rates by complex III were significantly lower than that seen with acetylated cytochrome c. The reduction of these modified cytochromes c under conditions where complex I generates superoxide was almost completely (about 90%) inhibited by superoxide dismutase. The genetically modified cytochromes c are useful analytical reagents for studies on superoxide generation by the mitochondrial respiratory chain. Quantitative comparison of superoxide-mediated cytochrome c reduction with hydrogen peroxide-mediated Amplex Red oxidation suggests that complex I within its native environment (submitochondrial particles) produces both superoxide (∼50%) and hydrogen peroxide (∼50%).
Keywords: cytochrome c ; site-directed mutagenesis; complex I; respiratory chain; superoxide radical; hydrogen peroxide; mitochondria

Resistance of α-crystallin quaternary structure to UV irradiation by A. V. Krivandin; K. O. Muranov; F. Yu. Yakovlev; N. B. Poliansky; L. A. Wasserman; M. A. Ostrovsky (633-642).
The damaging effect of UV radiation (λ > 260 nm) on bovine α-crystallin in solution was studied by small-angle X-ray scattering, gel permeation chromatography, electrophoresis, absorption and fluorescence spectroscopy, and differential scanning calorimetry. The results obtained show that damage to even a large number of subunits within an α-crystallin oligomer does not cause significant rearrangement of its quaternary structure, aggregation of oligomers, or the loss of their solubility. Due to the high resistance of its quaternary structure, α-crystallin is able to prevent aggregation of destabilized proteins (especially of γ- and β-crystallins) and so to maintain lens transparency throughout the life of an animal (the chaperone-like function of α-crystallin).
Keywords: α-crystallin; quaternary structure; UV radiation; small-angle X-ray scattering

Uncoupling by ammonium of electron transfer and ATP synthesis during linear transfer of electrons from water to photosystem 1 acceptors was studied in pea chloroplasts. It was shown that 40 μM palmitic acid decreased several-fold the ammonium concentrations necessary for 50% inhibition of ATP synthesis. The protonophore carbonyl cyanide m-chlorophenylhydrazone has no such property. The enhancement by palmitate of ammonium-induced uncoupling is accompanied by acceleration of basal electron transfer and decrease in the photoinduced uptake of hydrogen ions (H+). In the absence of ammonium, palmitate has no effect on basal transport and stimulates uptake of hydrogen ions. This means that in the case of combined action of palmitate and ammonium an additional leakage of H+ takes place, resulting in dissipation of the pH gradient. Synergic action of two metabolites, free fatty acid and ammonium, is supposed to provide for functioning of a system of mild regulation of energy coupling processes in native plant cell chloroplasts. Possible mechanisms of synergism are discussed.
Keywords: ammonium uncoupling; palmitic acid; electron transfer; ATP synthesis; chloroplasts

Probing for actinase activity of protealysin by O. A. Tsaplina; T. N. Efremova; L. V. Kever; Ya. Yu. Komissarchik; I. V. Demidyuk; S. V. Kostrov; S. Yu. Khaitlina (648-654).
The ability of protealysin, a thermolysin-like metallopeptidase from Serratia proteamaculans 94, to cleave actin and matrix metalloprotease MMP2 is reported. In globular actin, protealysin and S. proteamaculans 94 cell extracts are shown to hydrolyze the Gly42-Val43 peptide bond within the DNase-binding loop and the Gly63-Ile64 and Thr66-Ile67 peptide bonds within the nucleotide cleft of the molecule. At enzyme/substrate mass ratio of 1: 50 and below, a 36 kDa-fragment produced by the cleavage between Gly42 and Val43 was virtually resistant to further breakdown. Judging from the results of zymography, protealysin transforms proMMP2 into a 66 kDa polypeptide characteristic of mature MMP2, indicating that protealysin can activate MMP2. Upon incubation of S. proteamaculans 94 with human larynx carcinoma Hep-2 cells intracellular bacteria were detected in about 10% of Hep-2 cells, this being the first evidence for invasion of eukaryotic cells with bacteria of this species. Thus, S. proteamaculans 94 turned out to be one more bacterial strain in which synthesis of actin-specific metalloprotease is coupled with bacterial invasion. These results are consistent with the idea of the actinase activity of bacterial metalloproteases being a factor that may promote bacterial invasion of eukaryotic cells.
Keywords: actin; metalloprotease; Serratia ; bacterial invasion; ECP32; grimelysin

Enzymological properties of endo-(1–4)-β-glucanase Eg12p of Penicillium canescens and characteristics of structural gene egl2 by A. M. Chulkin; D. S. Loginov; E. A. Vavilova; A. R. Abyanova; I. N. Zorov; S. A. Kurzeev; O. V. Koroleva; S. V. Benevolenskii (655-662).
Gene egl2 of secreted endo-(1–4)-β-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding β-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60°C, respectively, exhibited specific activity of 33 IU, and had K m and V max in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 μmol/sec per mg, respectively.
Keywords: endoglucanase; mycelial fungus; Penicillium canescens ; gene

Safranine O as a fluorescent probe for mitochondrial membrane potential studied on the single particle level and in suspension by I. V. Perevoshchikova; A. I. Sorochkina; D. B. Zorov; Y. N. Antonenko (663-671).
The permeant cationic dye safranine O is often used to measure mitochondrial membrane potential due to the dependence of both its absorption and fluorescence on mitochondrial energization, which causes its oligomerization inside mitochondria. In the present study we have used fluorescent correlation spectroscopy (FCS) to record the fluorescence changes on a micro level, i.e. under conditions permitting resolution of contributions from single particles (molecules of the dye and stained mitochondria). We have shown that the decrease in fluorescence signal from a suspension of energized mitochondria stained with a high safranine concentration (10 μM) is explained by the decrease in dye concentration in the medium in parallel with the accumulation of the dye inside the mitochondria, which results in fluorescence quenching. With 1 μM safranine O, the fluorescence rise after energization is caused by the accumulation of the dye up to a level not sufficient for full fluorescence quenching and also by the higher intensity of mitochondrial fluorescence on immersion of the dye in the hydrophobic milieu. Besides the estimation of the inner mitochondrial membrane potential, this approach also assesses the concentration of fluorescent particles. The non-monotonic dependence of the FCS parameter 1/G(τ→0) on the concentration of mitochondrial protein suggests heterogeneity of the system with respect to fluorescence of particles. An important advantage of the described method is its high sensitivity, which allows measurements with low concentrations and quantities of mitochondrial protein in samples (less than 10 μg).
Keywords: mitochondria; safranine O; potential; fluorescence; FCS; nanoparticles; heterogeneity; suspension

Fusion of barnase to antiferritin antibody F11 VH domain results in a partially folded functionally active protein by D. V. Shubenok; Y. I. Tsybovsky; O. A. Stremovskiy; S. M. Deyev; S. P. Martsev (672-680).
A chimeric protein, VH-barnase, was obtained by fusing the VH domain of anti-human ferritin monoclonal antibody F11 to barnase, a bacterial RNase from Bacillus amyloliquefaciens. After refolding from inclusion bodies, the fusion protein formed insoluble aggregates. Off-pathway aggregation was significantly reduced by adding either purified GroEL/GroES chaperones or arginine, with 10–12-fold increase in the yield of the soluble protein. The final protein conformation was identical by calorimetric criteria and CD and fluorescence spectroscopy to that obtained without additives, thus suggesting that VH-barnase structure does not depend on folding conditions. Folding of VH-barnase resulted in a single calorimetrically revealed folding unit, the so-called “calorimetric domain”, with conformation consistent with a molten globule that possessed well-defined secondary structure and compact tertiary conformation with partial exposure of hydrophobic patches and low thermodynamic stability. The unique feature of VH-barnase is that, despite the partially unfolded conformation and coupling into a single “calorimetric domain”, this immunofusion retained both the antigen-binding and RNase activities that belong to the two heterologous domains.
Keywords: VH domain; barnase; protein folding; immunofusion; molten globule; differential scanning calorimetry

Sulfation of N-acyl dopamines in rat tissues by M. G. Akimov; I. V. Nazimov; N. M. Gretskaya; G. N. Zinchenko; V. V. Bezuglov (681-685).
Sulfation of N-acyl dopamines has been shown for the first time in cytosolic fractions of rat liver and nervous system. Sulfation of dopamine amides of docosahexaenoic and oleic acids occurred in all tissues studied, N-arachidonoyl dopamine was sulfated in the liver and spinal cord, and N-stearoyl dopamine was sulfated only in the liver. Depending on the substrate and tissue, the sulfation activity varied from 0.5 to 3.5 nmol/min per mg total protein. Kinetic parameters of N-docosahexaenoyl dopamine sulfation in the brain were determined. The findings characterize the sulfation system as the most productive metabolic pathway of N-acyl dopamines, but the role of this system in the body is unclear because of high K m value.
Keywords: aryl sulfotransferase; neurolipins; metabolism; liver; nervous system

Intracellular content of hydrogen peroxide and of the product of lipid peroxidation malonic dialdehyde as well as activity of antioxidant enzymes catalase, ascorbate peroxidase, and superoxide dismutase were studied in cells of morphogenic and derived from them non-morphogenic calluses of tatar buckwheat Fagopyrum tataricum L. Non-morphogenic calluses were characterized by significantly higher content of hydrogen peroxide and malonic dialdehyde, low catalase activity, and high activity of superoxide dismutase compared to morphogenic cultures. The results may indicate that cells of non-morphogenic calluses are in the state of continuous oxidative stress. Nevertheless, proliferative activity of non-morphogenic cultures and the biomass increase significantly exceeded these parameters in morphogenic calluses. An analogy is drawn between animal cancer cells and non-morphogenic plant calluses.
Keywords: morphogenic and non-morphogenic calluses; hydrogen peroxide; malonic dialdehyde; oxidative stress; antioxidant enzymes

Comparative study of immobilized and soluble NADH:FMN-oxidoreductase-luciferase coupled enzyme system by E. N. Esimbekova; I. G. Torgashina; V. A. Kratasyuk (695-700).
The properties of a coupled enzyme system (NAD(P)H:FMN-oxidoreductase and luciferase) from luminous bacteria were studied. The enzymes and their substrates were immobilized in polymer gels of different types: starch (polysaccharide) and gelatin (polypeptide). Maximum activity yield (100%) was achieved with the enzymes immobilized in starch gel. An increase in K m app was observed in both immobilized systems as compared with the soluble coupled enzyme system. Immobilization in starch and gelatin gels increased the resistance of the NAD(P)H:FMN-oxidoreductase and luciferase coupled enzyme system to the effects of external physical and chemical factors. The optimum pH range expanded both to the acidic and alkaline regions. The resistance to concentrated salt solutions and high temperature also increased. The coupled enzyme system immobilized in starch gel (with activation energy 30 kJ/mol) was characterized by the best thermostability. The immobilized coupled enzyme system can be used to produce a stable and highly active reagent for bioluminescent analysis.
Keywords: bioluminescence; immobilization; thermostability; luciferase; starch; gelatin

Experimental Glycoscience. Glycobiology by G. Ya. Wiederschain (701-702).