Biochemistry (Moscow) (v.73, #10)

Polygalacturonase-inhibiting protein is a structural component of plant cell wall by M. A. Protsenko; N. L. Buza; A. A. Krinitsyna; E. A. Bulantseva; N. P. Korableva (1053-1062).
It is generally believed that plants “evolved a strategy of defending themselves from a phytopathogen attack” during evolution. This metaphor is used frequently, but it does not facilitate understanding of the mechanisms providing plant resistance to the invasion of foreign organisms and to other unfavorable external factors, as well as the role of these mechanisms in plant growth and development. Information on processes involving one of the plant resistance factors—polygalacturonase-inhibiting protein (PGIP)—is considered in this review. The data presented here indicate that PGIP, being an extracellular leucine-rich repeat-containing protein, performs important functions in the structure of plant cell wall. Amino acid residues participating in PGIP binding to homogalacturonan in the cell wall have been determined. The degree of methylation and the mode of distribution of homogalacturonan methyl groups are responsible for the formation of a complex structure, which perhaps determines the specificity of PGIP binding to pectin. PGIP is apparently one of the components of plant cell wall determining some of its mechanical properties; it is involved in biochemical processes related to growth, expansion, and maceration, and it influences plant morphology. Polygalacturonase (PG) is present within practically all plant tissues, but the manifestation of its activity varies significantly depending on physiological conditions in the tissue. Apparently, the regulation of PG functioning in apoplast significantly affects the development of processes associated with the modification of the structure of plant cell wall. PGIP can regulate PG activity through binding to homogalacturonan. The genetically determined structure of PGIP in plants determines the mode of its interaction with an invader and perhaps is one of the factors responsible for the set of pathogens causing diseases in a given plant species.
Keywords: polygalacturonase-inhibiting protein; PGIP; polygalacturonase; homogalacturonan; plant cell wall; resistance; LRR proteins; pectin

Oxygen as an alternative electron acceptor in the photosynthetic electron transport chain of C3 plants by I. V. Kuvykin; A. V. Vershubskii; V. V. Ptushenko; A. N. Tikhonov (1063-1075).
This study deals with effects of oxygen on the kinetics of P700 photoinduced redox transitions and on induction transients of chlorophyll fluorescence in leaves of C3 plants Hibiscus rosa-sinensis and Vicia faba. It is shown that the removal of oxygen from the leaf environment has a conspicuous effect on photosynthetic electron transport. Under anaerobic conditions, the concentration of oxidized P700 centers in continuous white light was substantially lower than under aerobic conditions. The deficiency of oxygen released non-photochemical quenching of chlorophyll fluorescence, thus indicating a decrease in the trans-thylakoid pH gradient (ΔpH). Quantitative analysis of experimental data within the framework of an original mathematical model has shown that the steady-state electron flux toward oxygen in Chinese hibiscus leaves makes up to ∼40% of the total electron flow passing through photosystem 1 (PS1). The decrease in P 700 + content under anaerobic conditions can be due to two causes: i) the retardation of electron outflow from PS1, and ii) the release of photosynthetic control (acceleration of electron flow from PS2 to P 700 + ) owing to lower acidification of the intra-thylakoid space. At the same time, cyclic electron transport around PS1 was not stimulated in the oxygen-free medium, although such stimulation seemed likely in view of possible rearrangement of electron flows on the acceptor side of PS1. This conclusion stems from observations that the rates of P 700 + reduction in DCMU-poisoned samples, both under aerobic and anaerobic conditions, were negligibly small compared to rates of electron flow from PS2 toward P 700 + in untreated samples.
Keywords: C3 plants; chloroplasts; electron transport; EPR; non-photochemical fluorescence quenching; mathematical modeling

Reactive oxygen species in programmed death of pea guard cells by V. D. Samuilov; D. B. Kiselevsky; A. A. Shestak; A. V. Nesov; L. A. Vasil’ev (1076-1084).
Hydrogen peroxide potentiates CN-induced apoptosis of guard cells recorded as destruction of cell nuclei in the epidermis from pea leaves. A still stronger effect was exerted by the addition of H2O2 and NADH, which are the substrates of the plant cell wall peroxidase producing $$ O_2^{underset{ aise0.3emhbox{$smash{scriptscriptstylecdot}$}}{ - } } $$ coupled to the oxidation of NADH. The CN-or (CN + H2O2)-induced destruction of guard cell nuclei was completely removed by nitroblue tetrazolium (NBT) oxidizing $$ O_2^{underset{ aise0.3emhbox{$smash{scriptscriptstylecdot}$}}{ - } } $$ and preventing there-by the subsequent generation of H2O2. The reduced NBT was deposited in the cells as formazan crystals. Cyanide-induced apoptosis was diminished by mannitol and ethanol, which are OH· traps. The dyes Rose Bengal (RB) and tetramethylrhodamine ethyl ester (TMRE) photosensitizing singlet oxygen production suppressed the CN-induced destruction of the cell nuclei in the light. This suppression was removed by exogenous NADH, which reacts with 1O2 yielding $$ O_2^{underset{ aise0.3emhbox{$smash{scriptscriptstylecdot}$}}{ - } } $$ . Incubation of leaf slices with RB in the light lowered the photosynthetic O2 evolution rate and induced the permeability of guard cells for propidium iodide, which cannot pass across intact membranes. Inhibition of photosynthetic O2 evolution by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or bromoxynil prevented CN-induced apoptosis of guard cells in the light but not in the dark. RB in combination with exogenous NADH caused H2O2 production that was sensitive to NBT and estimated from dichlorofluorescein (DCF) fluorescence. Data on NBT reduction and DCF and TMRE fluorescence obtained using a confocal microscope and data on the NADH-dependent H2O2 production are indicative of generation of reactive oxygen species in the chloroplasts, mitochondria, and nuclear region of guard cells as well as with participation of apoplastic peroxidase. Cyanide inhibited generation of reactive oxygen species in mitochondria and induced their generation in chloroplasts. The results show that H2O2, OH·, and $$ O_2^{underset{ aise0.3emhbox{$smash{scriptscriptstylecdot}$}}{ - } } $$ resources utilized for H2O2 production are involved in apoptosis of guard cells. It is likely that singlet oxygen generated by RB in the light, judging from the permeability of the plasmatic membrane for propidium iodide, makes Photosystem II of chloroplasts inoperative and induces necrosis of the guard cells.
Keywords: programmed cell death; reactive oxygen species; chloroplasts; mitochondria; cell nucleus; apoplastic peroxidase; Pisum sativum L.

To study the interrelation between the spectral and structural properties of fluorescent proteins, structures of mutants of monomeric red fluorescent protein mRFP1 with all possible point mutations of Glu66 (except replacement by Pro) were simulated by molecular dynamics. A global search for correlations between geometrical structure parameters and some spectral characteristics (absorption maximum wavelength, integral extinction coefficient at the absorption maximum, excitation maximum wavelength, emission maximum wavelength, and quantum yield) was performed for the chromophore and its 6 environment in mRFP1, Q66A, Q66L, Q66S, Q66C, Q66H, and Q66N. The correlation coefficients (0.81–0.87) were maximal for torsion angles in phenolic and imidazolidine rings as well as for torsion angles in the regions of connection between these rings and chromophore attachment to β-barrel. The data can be used to predict the spectral properties of fluorescent proteins based on their structures and to reveal promising positions for directed mutagenesis.
Keywords: fluorescent proteins; mRFP1; mutants at residue 66; spectral characteristics; structure; correlations; molecular dynamics

In the present work we summarize results on construction of expression plasmid, heterologous expression in Escherichia coli, isolation and purification, as well as physicochemical characterization of chimeric protein consisting of hydrophilic domain of cytochrome b 5 and truncated from the N-terminal sequence (Δ23) form of NADH-cytochrome b 5 reductase. The kinetics and mechanism of electron transfer between NADH-cytochrome b 5 reductase and cytochrome b 5 in the frames of fusion protein consisting of cytochrome b 5 (94 amino acids) and truncated form of NADH-cytochrome b 5 reductase (277 amino acids) have been studied. It is shown that electron transfer takes place between redox partners belonging to two different molecules of the chimeric protein. Using computer modeling, we built the model of the tertiary structure of the fusion protein, which is in agreement with experimental data. By using Marcus theory of electron transfer in polar media, we demonstrate the inability of the hypothesis of electrostatic repulsions to explain the increase of electron transfer rate on increase of ion concentration in media due to elimination of the repulsion of similar charges. The real reason for the increase of the first order rate constant in some oxidation-reduction reactions between proteins, as shown in the present work, is a decrease of the media reorganization energy resulting in decrease of activation energy for oxidation-reduction reactions.
Keywords: microsomal cytochrome b 5 ; expression in Escherichia coli ; affinity chromatography; purification

Highly efficient expression of Escherichia coli heat-labile enterotoxin B subunit in plants using potato virus X-based vector by N. V. Ravin; V. V. Kuprianov; L. A. Zamchuk; A. V. Kochetov; Yu. L. Dorokhov; J. G. Atabekov; K. G. Skryabin (1108-1113).
A synthetic gene of the B-subunit of Escherichia coli heat-labile toxin, optimized for expression in plants, was designed and synthesized. The recombinant viral vector was constructed on the basis of potato virus X containing the LTB gene instead of the removed triple block of transport genes and the coat protein gene, which provides for LTB expression in plants. The vector is introduced into the plant cells during cell infiltration by agrobacteria incorporating a binary vector, the T-DNA region of which contains a cDNA copy of the recombinant viral genome. Under conditions of posttranscriptional gene silencing inhibition, the LTB yield in Nicotiana benthamiana plants is 1–2% of total soluble protein; in this case, LTB synthesized in plants forms pentameric complexes analogous to those found in the native toxin. The designed viral system of LTB transient expression can be used to obtain in plants a vaccine against enteropathogenic Escherichia coli.
Keywords: plant as “biofactories”; LTB; vaccine; viral vector

Steady-state kinetics of compulsory-ordered single-substrate irreversible and reversible enzyme reactions with two, three, and arbitrary number of intermediates were observed. Necessary and sufficient conditions for application of the quasi-equilibrium assumption and restrictions of this assumption were found in cases of two and three intermediates in the equilibrium segment. For all cases, accuracy of the quasi-equilibrium assumption was evaluated.
Keywords: enzyme kinetics; quasi-equilibrium assumption; rapid equilibrium; theory; steady-state kinetics; Cha method; accuracy of assumption

It is known that an addition of FeSO4 in the presence of ascorbic acid to cells or mitochondria can injure energy coupling and some other functions in mitochondria. The present study demonstrates that decrease in ascorbate concentration from 4 to 0.2 mM in the presence of the same low concentrations of FeSO4 accelerates the nonspecific pore opening, while cyclosporin A prevents and under some conditions reverses the pore opening. Hydrophobic cations SkQ1 and MitoQ (structural analogs of plastoquinone and coenzyme Q10, respectively) delay pore opening, SkQ1 being more efficient. It is known that an increase in matrix ADP concentration delays pore opening, while an addition of carboxyatractylate to mitochondria accelerates the beginning of pore opening. Preliminary addition of SkQ1 into a mitochondrial suspension increased the effect of ADP and decreased the effect of carboxyatractylate. These results suggest that under the conditions used SkQ1 protects mitochondria from oxidative damage as an antioxidant when added at extremely low concentrations.
Keywords: mitochondria; nonspecific Ca2+-dependent cyclosporin A-sensitive pore; oxidative stress; SkQ1; MitoQ; antioxidants

Isolation and properties of collagenolytic serine proteinase isoenzyme from king crab Paralithodes camtschatica by S. A. Semenova; G. N. Rudenskaya; L. V. Lyutova; O. A. Nikitina (1125-1133).
An electrophoretically homogeneous isoenzyme CSP-2 of collagenolytic serine proteinase has been isolated from the total preparation of king crab digestive enzymes. The molecular mass of the proteinase is 24.8 ± 0.3 kD, pH optimum for activity is 8.5, the temperature optimum for activity is 38–40°C, and the pH range of stability is 7–10. The enzyme has dual substrate specificity, but preference for positively charged amino acid residues in P1-position is more pronounced than in the case of the major isoenzyme. The temperature dependence of kinetic constants for synthetic substrate hydrolysis by CSP-2 has been investigated. Inhibition specificity of the enzyme is characteristic of serine proteinases but more like that of crab trypsin than that of the major CSP isoenzyme. The isolated collagenolytic proteinase also cleaves fibrinogen and fibrin and activates plasminogen. The amino acid sequence of the CSP-2 proteinase, which has been partially determined by tandem mass spectrometry, displays some similarity to the sequence of the major CSP isoenzyme.
Keywords: king crab; psychrophilic proteinase; kinetic constants; collagenolysis; fibrinolysis

Latent membrane protein 1 (LMP1) of the Epstein-Barr virus is a constitutively activated analog of the tumor necrosis factor receptor TNF-R1. LMP1 serves as a viral oncogen able to transform human B-ymphocytes and rodent fibroblasts via activation of numerous cellular signal cascades. Two specific motifs within LMP1 are responsible for interaction of this viral protein with the receptor protein β-TrCP/HOS SCF of the ubiquitin ligase E3 complex, playing an important role in degradation of numerous cellular proteins including NF-κB inhibitor IκBα. In this study, we demonstrate for the first time the importance of point mutations affecting HOS-recognizing motifs of LMP1 for activation of NF-κB, AP1, and PI3K/Akt signaling pathways. It has also been shown that rat fibroblast cell lines (Rat-1) expressing different HOS mutants of LMP1 produce different amounts of reactive nitrogen species. Our data confirm the hypothesis that point mutations in the C-terminal region of the LMP1 cytoplasmic domain can influence the transforming potential of the Epstein-Barr virus.
Keywords: Epstein-Barr virus; LMP1 protein; HOS mutations; NF-κB; NO

New determinants of Thermoactinomyces vulgaris carboxypeptidase T (CPT) substrate specificity—structural calcium ions and Leu254 residue—were found by means of steady-state kinetics and site-directed mutagenesis. The removal of calcium ions shifted the selectivity profile of hydrolysis of tripeptide substrates with C-terminal Leu, Glu, and Arg from 64/1.7/1 to 162/1.3/1. Substitution of the hydrophobic Leu254 in CPT for polar Asn did not change hydrolysis efficiency of substrates with C-terminal Leu and Arg, but resulted in more than 28-fold decrease in activity towards the substrate with C-terminal Glu. It is shown that the His68 residue is not a structural determinant of CPT specificity.
Keywords: carboxypeptidase T; substrate specificity; rational redesign; protein engineering; site-directed mutagenesis

Calcium uptake in rat liver mitochondria accompanied by activation of ATP-dependent potassium channel by O. V. Akopova; V. I. Nosar; I. N. Mankovskaya; V. F. Sagach (1146-1153).
The influence of potassium ions on calcium uptake in rat liver mitochondria is studied. It is shown that an increase in K+ and Ca2+ concentrations in the incubation medium leads to a decrease in calcium uptake in mitochondria together with a simultaneous increase in potassium uptake due to the potential-dependent transport of K+ in the mito-chondrial matrix. Both effects are more pronounced in the presence of an ATP-dependent K+-channel (K ATP + -channel) opener, diazoxide (Dz). Activation of the K ATP + -channel by Dz alters the functional state of mitochondria and leads to an increase in the respiration rate in state 2 and a decrease in the oxygen uptake and the rate of ATP synthesis in state 3. The effect of Dz on oxygen consumption in state 3 is mimicked by valinomycin, but it is opposite to that of the classical protonophore uncoupler CCCP. It is concluded that the potential-dependent uptake of potassium is closely coupled to calcium transport and is an important parameter of energy coupling responsible for complex changes in oxygen consumption and Ca2+-transport properties of mitochondria.
Keywords: ATP-dependent K+-channels; mitochondria; potassium; calcium; transport; oxygen consumption; diazoxide

Isolation and characterization of Lentinus edodes (Berk.) singer extracellular lectins by O. M. Tsivileva; V. E. Nikitina; E. A. Loshchinina (1154-1161).
Lectin preparations have been isolated and purified from the culture liquid of the xylotrophic basidiomycete Lentinus edodes (Berk.) Singer [Lentinula edodes (Berk.) Pegler]. The culture of L. edodes F-249 synthesizes two extracellular lectins different in composition and physicochemical properties. Extracellular lectin L1 from L. edodes is a glycoprotein of mono-subunit structure with molecular weight of 43 kD. L1 is comprised of 10.5 ± 1.0% (w/w) carbohydrates represented by glucose (Glc). Extracellular lectin L2 is a proteoglycan of mono-subunit structure with molecular weight of 37 kD. L2 is comprised of 90.3 ± 1.0% (w/w) carbohydrates represented by Glc (73% of the total mass of the carbohydrate moiety of the lectin molecule) and galactose (Gal) (27% of the total mass of the carbohydrate part of the lectin molecule). The content of Asn in L2 is high, i.e. 42% (w/w) of total amino acids. This fact along with the composition of the carbohydrate part of the molecule (Glc + Gal) allows one to assign L2 to N-asparagine-bound proteins. Both lectins are specific to D-Gal and lactose (Lac) at an equal for L1 and L2 minimal inhibiting concentration of these carbohydrates (2.08 mM Gal and 8.33 mM Lac). Other carbohydrates to which the lectins show affinity are different for the two lectins: Rha (4.16 mM) for L1 and Ara (4.16 mM) and mannitol (8.33 mM) for L2. The purified extracellular lectins of L. edodes are highly selective at recognition of definite structures on the surface of trypsinized rabbit erythrocytes and do not react with the erythrocytes of other animals and humans.
Keywords: Lentinus edodes ; lectins of higher fungi; extracellular glycoproteins; protein isolation and purification

DNA condensation plays a key role in non-viral gene delivery by affecting gene transfection, nuclear targeting, and eventual gene expression efficiency. Theoretically, a DNA condenser with the appropriate DNA condensation ability but without affecting DNA dissociation from DNA condensates inside the cytoplasm should be a perfect carrier for gene delivery. Protamine is a natural DNA condensation agent and has been widely used in gene delivery. In this work, protamine was selectively digested enzymatically to produce low molecular weight protamine fragments (LMWPs) of various lengths and amino acid compositions. The DNA condensation ability and gene transfection efficiency of these LMWP peptides were tested. Compared to protamine, all the LMWP peptides showed lower DNA binding strength. However, some LMWP peptides demonstrated excellent DNA condensation ability and could form very compact DNA condensates with small particle size (∼100 nm). More interestingly, LMWP peptide-mediated in vitro gene delivery showed prolonged (up to 12 days) gene expression. Results from this study suggest that designing DNA condensers with appropriate and tunable DNA binding strengths and condensation abilities would be an effective means to improve gene expression and thus gene therapy efficiency. Since LMWP peptides have low immunogenicity, they would be safer than protamine for use in gene therapies.
Keywords: peptide; enzyme digestion; DNA condensation; controlled gene expression

Affinity Chromatography. Methods and Protocols by G. Ya. Wiederschain (1169-1169).