Biochemistry (Moscow) (v.75, #11)

Transcription Factories and Spatial Organization of Eukaryotic Genomes by S. V. Razin; A. A. Gavrilov; O. V. Yarovaya (1307-1315).
The phenomenon of association of transcribed genes into so-called transcription factories and also the role of these associations in spatial organization of the eukaryotic genome are actively discussed in the modern literature. Some authors think that the association of transcribed genes into transcription factories constitutes a major factor supporting the function-dependent three-dimensional organization of the interphase genome. In spite of the obvious interest in the prob- lem of spatial organization of transcription in the eukaryotic cell nucleus, the number of experimental studies of transcrip- tional factories remains rather limited and the results of these studies are often contradictory. In the current review we have tried to critically re-evaluate the published experimental results that constitute the basis for current models and also the models themselves. We have especially analyzed the existing contradictions and attempted to explain them whenever possi- ble. We also discuss new models that can explain the biological significance of clustering of transcribed genes and show pos- sible mechanisms of the origin of transcription factories in the course of evolution.
Keywords: genome spatial organization; immobilized RNA polymerase; nuclear matrix; remote gene interaction; transcriptional interaction; regulation of gene expression

A-to-I RNA Editing: A Contribution to Diversity of the Transcriptome and an Organism’s Development by A. A. Jr Zamyatnin; K. G. Lyamzaev; R. A. Zinovkin (1316-1323).
The complexity of multicellular organisms requires both an increase in genetic information and fine tuning in regulation of gene expression. One of the mechanisms responsible for these functions is RNA editing. RNA editing is a complex process affecting the mechanism of changes in transcriptome sequences. The best studied example of this process is A-to-I RNA editing. On the organism’s level, RNA editing plays a key role during ontogenesis and in the defense against pathogens. Disorders in A-to-I RNA editing lead to serious abnormalities. The importance of RNA editing increases with an increase in the organism’s complexity. Correct RNA editing is an indispensable factor of an organism’s development and probably determines the lifespan of higher eukaryotes.
Keywords: ADAR1; ADAR2; microRNA; RNA interference; alternative splicing; translational regulation

Study of α-Crystallin Structure by Small Angle Neutron Scattering with Contrast Variation by A. V. Krivandin; T. N. Murugova; A. I. Kuklin; K. O. Muranov; N. B. Poliansky; V. L. Aksenov; M. A. Ostrovsky (1324-1330).
The structure of the oligomeric protein α-crystallin from bovine eye lens was investigated by small-angle neutron scattering (SANS) with contrast variation. Based on the SANS curves, the match point for α-crystallin (43% D2O) and its average scattering length density at this point (2.4•1010 cm-2) were evaluated. The radius of gyration and the distance distri- bution functions for α-crystallin were calculated. On the basis of these calculations, it was concluded that α-crystallin is characterized by homogeneous distribution of scattering density in the domains inaccessible for water penetration, and all polypeptide subunits in α-crystallin oligomers undergo equal deuteration. The latter indicates that all α-crystallin subunits are equally accessible for water and presumably for some other low molecular weight substances. These conclusions on the α-crystallin structure (homogeneous distribution of scattering density and equal accessibility of all subunits for low molecular weight substances) should be taken into account when elaborating a-crystallin quaternary structure models.
Keywords: α-crystallin; quaternary structure; small-angle neutron scattering; contrast variation

Influence of Distamycin, Chromomycin, and UV-irradiation on Extraction of Histone H1 from Rat Liver Nuclei by Polyglutamic Acid by A. N. Prusov; T. A. Smirnova; L. P. Kurochkina; G. Ya. Kolomijtseva (1331-1341).
Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A3 (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this dif- ference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and s~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (s~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV- stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (s~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.
Keywords: cell nucleus; chromatin; histone H1; polyglutamic acid; distamycin; chromomycin; UV irradiation

Changes in phospholipid composition, phospholipase activity, and accumulation of lipid peroxidation products in mycelium of the lignindegrading fungus Lentinus (Panus) tigrinus VKM F-3616D in the presence of phenol and lignocellulosic substrates in the cultivation medium are reported. It is shown that in fungal mycelium in the presence of both substrates the share of lysophosphatidylcholine sharply increases. The parity between separate groups of phosphatidylinositols also changes. The lysophosphatidylcholine content increase during cultivation is connected with activation of phospholipase A2 (EC 3.1.1.4), and phosphatidylinositol parity change is associated with distinctions in affinity of phosphoinositidespecific phospholipase C (EC 3.1.4.11) to them.
Keywords: Lentinus tigrinus; phospholipids; phospholipase; xenobiotics; lignin; biodegradation

Catalase Activity of Cytochrome c Oxidase Assayed with Hydrogen Peroxide-Sensitive Electrode Microsensor by I. A. Bolshakov; T. V. Vygodina; R. Gennis; A. A. Karyakin; A. A. Konstantinov (1352-1360).
An iron-hexacyanide-covered microelectrode sensor has been used to continuously monitor the kinetics of hydrogen peroxide decomposition catalyzed by oxidized cytochrome oxidase. At cytochrome oxidase concentration ≈1 μM, the catalase activity behaves as a first order process with respect to peroxide at concentrations up to ≈300–400 μM and is fully blocked by heat inactivation of the enzyme. The catalase (or, rather, pseudocatalase) activity of bovine cytochrome oxi- dase is characterized by a second order rate constant of ≈2•102 M-1•sec-1 at pH 7.0 and room temperature, which, when divided by the number of H2O2 molecules disappearing in one catalytic turnover (between 2 and 3), agrees reasonably well with the second order rate constant for H2O2-dependent conversion of the oxidase intermediate FI-607 to FII-580. Accordingly, the catalase activity of bovine oxidase may be explained by H2O2 procession in the oxygen-reducing center of the enzyme yielding superoxide radicals. Much higher specific rates of H2O2 decomposition are observed with preparations of the bacterial cytochrome c oxidase from Rhodobacter sphaeroides. The observed second order rate constants (up to ≈3000 M-1•sec-1) exceed the rate constant of peroxide binding with the oxygen-reducing center of the oxidized enzyme (≈500 M-1•sec-1) several-fold and therefore cannot be explained by catalytic reaction in the a 3/CuB site of the enzyme. It is proposed that in the bacterial oxidase, H2O2 can be decomposed by reacting with the adventitious transition metal ions bound by the polyhistidine-tag present in the enzyme, or by virtue of reaction with the tightly-bound Mn2+, which in the bacterial enzyme substitutes for Mg2+ present in the mitochondrial oxidase.
Keywords: microsensor electrode; Prussian blue; respiratory chain; cytochrome c oxidase; oxygen intermediates; catalase activity

Study of Interaction of Ceruloplasmin with Serprocidins by V. Sokolov; K. V. Ageeva; V. A. Kostevich; M. N. Berlov; O. L. Runova; E. T. Zakharova; V. B. Vasilyev (1361-1367).
This paper describes formation of complexes of ceruloplasmin (CP) with such proteins of the serprocidin family as azurocidin (CAP37), neutrophilic elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). We present evidence that serprocidins form complexes with CP at a molar ratio 1: 1. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, did not prevent the interaction of serprocidins with CP in the course of SDS-free disc electrophoresis. CP affected the activities of NE, CG, and PR3 as a competitive inhibitor with K i ≈ 1 μM. Inhibitory effect of CP depended on ionic strength of the solution and was negligible at NaCl concentrations above 300 mM. In the mode of competitive inhibitors serprocidins suppressed oxidase activity of CP towards p-phenylenediamine. CAP37 displayed the strongest inhibitory effect (K i ≈20 nM). Upon adding various serprocidins to human, rat, rabbit, dolphin, dog, horse, and mouse plasma only CAP37 would form a complex with CP. Synthetic peptide RKARPRQFPRRR (5–13, 61–63 CAP37) displaced CAP37 from its complex with CP. Adding CAP37 to the triple complex formed by CP, lactoferrin, and myeloperoxidase resulted in displacement of the latter from the complex. The dissociation constant of CAP37 with immobilized CP was 13 nM. Therefore, among serprocidins CAP37 can be regarded as the specific partner of CP.
Keywords: ceruloplasmin; azurocidin; neutrophil elastase; cathepsin G; proteinase 3; protein-protein interactions

For the cyclooxygenase reaction of prostaglandin-H-synthase isolated from ram vesicular glands, dependences of the initial reaction rate, the maximal yield of the product, and the rate constant of enzyme inactivation in the course of reac- tion on oxygen concentration were studied in the absence and in the presence of electron donor in the reaction medium. It is shown that in the absence of electron donor the cyclooxygenase reaction is strictly governed by Michaelis-Menten kinet- ics over a wide range of oxygen concentrations (5–800 μM). In the presence of electron donor in the reaction medium it was found that cyclooxygenase reaction is inhibited by an excess of dissolved oxygen: the maximal values of the initial reaction rate and yield of the product are attained at oxygen concentration 50 μM, and its increase to 500 μM causes twofold decrease in the initial rate and maximal yield. The rate constant of enzyme inactivation in the course of reaction increases on increase in oxygen concentration both in the presence and in the absence of electron donor.
Keywords: prostaglandin-H-synthase; cyclooxygenase activity; kinetic mechanism; oxygen; substrate inhibition; hexa-cyanoferrate(II)

Application of the quasi-equilibrium assumption for the steady-state kinetics of bisubstrate irreversible enzymat- ic reactions in the case of ordered binding of substrates (Bi-Bi ordered mechanism) is considered. The necessary and suffi- cient conditions for application of the quasi-equilibrium assumption have been found and accuracy of this assumption has been numerically evaluated. The limitations on application of the quasi-equilibrium assumption have been shown and errors of its application have been analyzed. It is shown that possible discrimination of substrate binding order using asymmetrical expressions grounded on the quasi-equilibrium assumption is inconsistent because such asymmetrical expressions arise from incorrect application of the quasi-equilibrium assumption. Moreover, it has been proved in the general case that mecha- nisms generating such substrate-asymmetrical expressions for the steady-state rate of enzymatic reaction do not exist. The error source when using graphical interpretation for discrimination of mechanisms of bisubstrate enzymatic reactions has been determined. The strategy to avoid such errors is pointed out.
Keywords: enzymatic kinetics; bisubstrate reactions; quasi-equilibrium assumption; Cha method; rapid equilibrium; assumption accuracy; steady-state kinetics; graph method; discrimination of mechanisms

Effect of Plastoquinone Derivative 10-(6′-Plastoquinonyl)decyltriphenylphosphonium (SkQ1) on Contents of Steroid Hormones and NO Level in Rats by V. A. Chistyakov; V. A. Serezhenkov; A. A. Alexandrova; N. P. Milyutina; V. N. Prokof’ev; E. V. Mashkina; L. V. Gutnikova; S. V. Dem’yanenko (1383-1387).
Introduction of the plastoquinone derivative 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) into male Wistar rats once a day for two weeks in doses of 25 and 250 nmol/kg led to elevation of 17ß-estradiol level in blood serum by 33 and 41%, respectively. At the same time, nitrate and nitrite contents in the rat blood serum increased by 49 and 34%, respectively. ESR spectroscopy with diethyldithiocarbamate-iron complex as a spin trap showed more than twofold increase in NO production in lungs, but not in blood, liver, and intestines, following the SkQ1 daily introduction at a dose of 25 nmol/kg.
Keywords: SkQ1; penetrating cations; steroid hormones; estradiol; nitrate; nitrite; nitric oxide

Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide bonds (Cys44–Cys89 and Cys139–Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only 4–5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating that the second disulfide bond (Cys139–Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44–Cys89) is not required.
Keywords: Delonix regia trypsin inhibitor; Kunitz-type trypsin inhibitor; Delonix regia; reactive site; site-directed mutagenesis

Interaction of Plum Pox Virus with Specific Colloidal Gold-Labeled Antibodies and Development of Immunochromatographic Assay of the Virus by N. A. Byzova; I. V. Safenkova; S. N. Chirkov; V. G. Avdienko; A. N. Guseva; I. V. Mitrofanova; A. V. Zherdev; B. B. Dzantiev; J. G. Atabekov (1393-1403).
Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in sam- ples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by “sandwich”-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.
Keywords: plum pox virus; monoclonal antibodies; colloidal gold; transmission electron microscopy; atomic force microscopy; immunochromatographic assay

Properties of Partially Purified Endopolyphosphatase of the Yeast Saccharomyces cerevisiae by L. P. Lichko; T. V. Kulakovskaya; I. S. Kulaev (1404-1407).
Partially purified endopolyphosphatase from cytosol of the yeast Saccharomyces cerevisiae with inactivated genes PPX1 and PPN1 encoding exopolyphosphatases was obtained with ion_exchange and affinity chromatography. The enzyme activity was estimated by decrease of polyphosphate chain length determined by PAGE. The enzyme cleaved inorganic polyphosphate without the release of orthophosphate (Pi) and was inhibited by heparin and insensitive to fluoride. Mg2+, Mn2+, and Co2+ (1.5 mM) stimulated the activity, and Ca2+ was ineffective. The molecular mass of the endopolyphosphatase determined by gel filtration was of ≈20 kDa.
Keywords: inorganic polyphosphates; endopolyphosphatase; exopolyphosphatase; cytosol; PPX1 and PPN1 mutants; Saccharomyces cerevisiae

The Na,K-ATPase activity in microsomal fraction isolated from kidneys of winter hibernating ground squirrels was found to be 1.8–2.0-fold lower than that in active animals in summer. This is partially connected with a decrease in Na,K-ATPase protein content in these preparations (by 25%). Using antibodies to different isoforms of Na,K-ATPase α-subunit and analysis of enzyme inhibition by ouabain, it was found that the decrease in Na,K-ATPase activity during hibernation is not connected with change in isoenzyme composition. Seasonal changes of Na,K-ATPase a-subunit phosphory- lation level by endogenous protein kinases were not found. Proteins which could be potential regulators of Na,K-ATPase activity were not found among phosphorylated proteins of the microsomes. Analysis of the composition and properties of the lipid phase of microsomes showed that the total level of unsaturation of fatty acids and the lipid/protein ratio are not changed significantly during hibernation, whereas the cholesterol content in preparations from kidneys of hibernating ground squirrels is approximately twice higher than that in preparations from kidneys of active animals. However, using spin and fluorescent probes it was shown that this difference in cholesterol content does not affect the integral membrane micro-viscosity of microsomes. Using the cross-linking agent cupric phenanthroline, it was shown that Na,K-ATPase in mem- branes of microsomes from kidneys of hibernating ground squirrels is present in more aggregated state in comparison with membranes of microsomes from kidneys of active animals. We suggest that the decrease in Na,K-ATPase activity in kidneys of ground squirrels during hibernation is mainly connected with the aggregation of proteins in plasma membrane.
Keywords: hibernation; Na,K-ATPase; membrane microviscosity; protein aggregation; kidney; ground squirrel Spermophilus undulatus

Functional Glycomics by G. Ya. Wiederschain (1417-1417).

Handbook of Biochemistry and Molecular Biology by G. Ya. Wiederschain (1418-1418).