Biochemistry (Moscow) (v.74, #5)

Molecular bases of immune complex pathology by K. V. Shmagel; V. A. Chereshnev (469-479).
The binding of antigens with antibodies forms immune complexes in the body. Usually these complexes are eliminated by the system of mononuclear phagocytes without development of pathological changes. This review highlights principal mechanisms responsible for safe removal of immune complexes in primates and humans. Special attention is given to diseases known as “immune complex diseases”, when antigen-antibody complexes induce inflammatory reactions. The review considers key experimental works that significantly contributed to current knowledge of etiology and pathogenesis of type III hypersensitivity. Some factors of the development of immune complex syndrome such as level of humoral immune response to antigen, isotype and affinity of forming antibodies, the amount of immune complexes, and the consequences of their interaction with the complement system and Fc-receptors are analyzed based on the molecular mechanisms involved. The review contains a retrospective analysis of the most significant scientific achievements in immune complex pathology investigation within the last 100 years.
Keywords: immune complexes; complement; type III hypersensitivity; CR1 receptors; Fc-receptors; inflammation

Proteome analysis of the moss Physcomitrella patens (Hedw.) B.S.G. by A. Yu. Skripnikov; N. B. Polyakov; E. V. Tolcheva; V. V. Velikodvorskaya; S. V. Dolgov; I. A. Demina; M. A. Rogova; V. M. Govorun (480-490).
The sequencing of the moss Physcomitrella patens genome has facilitated studies of the plant proteome. To develop a proteome reference map based on the genome sequence, we conducted 2D electrophoreses of proteins extracted from moss protoplasts, protonemata, and gametophores grown under standard conditions on Petri dishes. On silver-stained gels, depending on the developmental stage of the moss, we resolved from 500 to 600 protein spots that were then excised and digested by trypsin, and 212 proteins were identified by PMF-MALDI-TOF. To enhance the proteome coverage, we performed 1D SDS-PAGE with subsequent separation of tryptic peptides derived from digested gel band slices by LC-ESI-MS/MS. The proposed approach allowed us to identify 186 proteins had not been determined by 2D PMF-MALDI-TOF. Proteins identified by both methods were categorized using a system of clusterization of orthologous genes as metabolism (26%), cellular processes and signaling (16%), and information storage and processing (7%). Proteome analysis by differential gel electrophoresis revealed moderate differences between filamentous protonemata and leafy shoots. Surprisingly, protoplasts isolated from protonema filaments displayed significant differences in protein composition compared with both protonemata and gametophores.
Keywords: proteomics; 2D electrophoresis; MALDI-TOF; LC-ESI-MS/MS; Physcomitrella patens ; protoplasts; DiGE

Photoactivated DNA analogs of nucleotide excision repair (NER) substrates have been created that are 48-mer duplexes containing in internal positions pyrimidine nucleotides with bulky substituents imitating lesions. Fluorochloroazidopyridyl, anthracenyl, and pyrenyl groups introduced using spacer fragments at 4N and 5C positions of dCMP and dUMP were used as model damages. The gel retardation and photo-induced affinity modification techniques were used to study the interaction of modified DNA duplexes with proteins in HeLa cell extracts containing the main components of NER protein complexes. It is shown that the extract proteins selectively bind and form covalent adducts with the model DNA. The efficiency and selectivity of protein modification depend on the structure of used DNA duplex. Apparent molecular masses of extract proteins, undergoing modification, were estimated. Mutual influence of simultaneous presence of extract proteins and recombinant NER protein factors XPC-HR23B, XPA, and RPA on interaction with the model DNA was analyzed. The extract proteins and RPA competed for interaction with photoactive DNA, mutually decreasing the yield of modification products. In this case the presence of extract proteins at particular concentrations tripled the increase in yield of covalent adducts formed by XPC. It is supposed that the XPC subunit interaction with DNA is stimulated by endogenous HR23B present in the extract. Most likely, the mutual effect of XPA and extract proteins stimulating formation of covalent adducts with model DNA is due to the interaction of XPA with endogenous RPA of the extract. A technique based on the use of specific antibodies revealed that RPA present in the extract is a modification target for photoactive DNA imitating NER substrates.
Keywords: DNA duplexes with bulky substituents; protein factors of pre-incision NER complexes; photoinduced affinity modification

The entire encoding region for Momordica charantia phospholipid hydroperoxide glutathione peroxidase (McPHGPx) was cloned into pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The purified recombinant McPHGPx displayed GSH-dependent peroxidase activity towards phospholipid hydroperoxide, H2O2, and tert-butyl hydroperoxide and had the highest affinity with and catalytic efficiency for phospholipid hydroperoxide. The optimum temperature of the enzyme activity ranged from 40 to 50°C, thus it is a thermostable enzyme compared to other PHGPx enzymes. Furthermore, McPHGPx expression in Saccharomyces cerevisiae PHGPx-deletion mutant rescued the susceptibilities to the oxidation-sensitive polyunsaturated fatty acid (linolenic acid), indicating its PHGPx complementation function in yeast. These results have well documented that McPHGPx functions as a PHGPx in vitro and in vivo and will be beneficial for further functional studies on plant PHGPx enzymes.
Keywords: phospholipid hydroperoxide glutathione peroxidase; Momordica charantia ; enzymatic properties; functional complementation; antioxidant enzyme

Manganese-dependent carboanhydrase activity of photosystem II proteins by A. V. Shitov; O. V. Pobeguts; T. N. Smolova; S. I. Allakhverdiev; V. V. Klimov (509-517).
Four sources of carbonic anhydrase (CA) activity in submembrane preparations of photosystem II (PS II) isolated from pea leaves were examined. Three of them belong to the hydrophilic proteins of the oxygen-evolving complex of PS II with molecular mass 33 kDa (protein PsbO), 24 kDa (protein PsbP), and 18 kDa (protein PsbQ). The fourth source of CA activity is associated with a pigment-protein complex of PS II after removing three hydrophilic proteins by salt treatment. Except for protein PsbQ, the CA activity of all these proteins depends on the presence of Mn2+: the purified protein PsbO did not show CA activity before adding Mn2+ into the medium (concentration of Mn2+ required for 50% effect, EC50, was 670 μM); CA activity of protein mixture composed of PsbP and PsbQ increased more than 5-fold upon adding Mn2+ (EC50 was 45 μM). CA activity of purified protein PsbP increased 2-fold in the presence of 200 μM Mn2+. As indicated for the mixture of two proteins (PsbP and PsbQ), Mg2+, Ca2+, and Zn2+, in contrast to Mn2+, suppressed CA activity (both initial and Mn2+-induced activity). Since the found sources of CA activity demonstrated properties different from ones of typical CA (need for Mn2+, insensitivity or low sensitivity to acetazolamide or ethoxyzolamide) and such CA activity was found only among PS II proteins, we cannot exclude that they belong to the type of Mn-dependent CA associated with PS II.
Keywords: photosystem II; carbonic anhydrase activity; oxygen-evolving complex; hydrophilic proteins of PS II-PsbO; PsbP; PsbQ

The conformational stabilities of chimeric protein Hmwb5-EGFP and its constituents (cytochrome b 5 and enhanced green fluorescent protein) in guanidine hydrochloride solutions are reported in this paper. Intensity of fluorescence of tryptophan residues, intensity of EGFP fluorescence in the visible region, absorbance of cytochrome b 5 heme and EGFP fluorophore, and fluorescence anisotropy were used to follow the unfolding process. Thermodynamic parameters of protein unfolding were obtained using different approaches. The data were analyzed using a two-stage model and a linear extrapolation method. Unfolding of protein molecules was additionally monitored by measuring Stern-Volmer constants for tryptophan fluorescence quenching by acrylamide, cesium, and iodide. The accessibility of tryptophan residues of both components in the fusion molecule is lower than in the separate molecules. The thermodynamic stability of the protein globules in the fusion protein is much lower than in the individual protein molecules in solution, the difference in free energy of unfolding being more considerable for cytochrome b 5 (29 ± 4 and 13 ± 2 kJ/mol) than for EGFP (26 ± 0.9 and 20 ± 2.7 kJ/mol). The data indicate that artificial protein fusion can greatly affect total structural stability, and in the case of cytochrome b 5 and EGFP it results in decrease in free energy of transition from native to denatured unfolded form and consequently to decrease in thermodynamic stability of protein globules compared to the separate proteins.
Keywords: microsomal cytochrome b 5 ; green fluorescent protein; fluorescence; folding; conformational stability

Physicochemical and kinetic characteristics of isoforms of isocitrate lyase from corn by A. T. Eprintsev; E. V. Maslova; D. N. Fedorin; V. N. Popov (528-532).
Three electrophoretically homogeneous isocitrate lyase (ICL) isoforms were obtained by 4-step purification from corn scutellum (ICL1 and ICL2) and green leaves (ICL). Their physicochemical, kinetic, and regulatory properties were analyzed. The molecular masses of ICL1, ICL2, and ICL isoforms determined by gel filtration are 164, 207, and 208 kDa, respectively. The proteins have homotetrameric quaternary structure with subunit molecular masses of 43, 48, and 47 kDa for ICL1, ICL2, and ICL, respectively. We found some differences in pH optimum, K m, and regulation by divalent metal cations between ICL1 and ICL2 and significant similarity of ICL2 and ICL. Based on these data, we suggest the participation of these isoforms in metabolic regulation of the glyoxylate cycle, organic acid metabolism during photorespiration in leaves and acidosis in corn seeds.
Keywords: isocitrate lyase; isoforms; electrophoresis; quaternary structure; subunits; glyoxylate cycle; gluconeogenesis

Polysaccharides of basidiomycetes. Alkali-soluble polysaccharides from the mycelium of white rot fungus Ganoderma lucidum (Curt.: Fr.) P. Karst by M. S. Evsenko; A. S. Shashkov; A. V. Avtonomova; L. M. Krasnopolskaya; A. I. Usov (533-542).
Two polysaccharides were isolated from submergedly cultured mycelium of the basidiomycete Ganoderma lucidum by extraction with alkali followed by fractionation with Fehling reagent. The polysaccharides were shown to be a linear (1→3)-α-D-glucan and a highly branched xylomannan containing a backbone built up of (1→3)-linked α-D-mannopyranose residues, the majority of which are substituted at O-4 by single β-D-xylopyranose residues or by disaccharide fragments β-D-Manp-(1→3)-β-D-Xylp-(1→. Polysaccharide structures were elucidated by NMR spectroscopy in combination with methylation analysis and periodate oxidation. An interesting feature of the xylomannan is the simultaneous presence of α-D-mannopyranose and β-D-mannopyranose residues, the first forming the backbone, and the second being the non-reducing terminal units of disaccharide side chains.
Keywords: Basidiomycetes; white rot fungi; Ganoderma lucidum ; polysaccharides; (1→3)-α-D-glucan; branched xylomannan; NMR spectroscopy

Anionic polymers of the cell wall of Bacillus subtilis subsp. subtilis VKM B-501T by A. S. Shashkov; N. V. Potekhina; S. N. Senchenkova; E. B. Kudryashova (543-548).
Teichoic acid and disaccharide-1-phosphate polymer were identified in the cell walls of Bacillus subtilis subsp. subtilis VKM B-501T. The teichoic acid represents 1,3-poly(glycerol phosphate) 80% substituted by α-D-glucopyranose residues at O-2 of glycerol. The linear repeating unit of disaccharide-1-phosphate polymer contains the residues of β-D-glucopyranose, N-acetyl-α-D-galactosamine, and phosphate and has the following structure: -6)-β-D-Glcp-(1→3)-α-D-GalpNAc-(1-P-. The structures of two anionic polymers were determined by chemical and NMR-spectroscopic methods. The 1H- and 13C-NMR spectral data on disaccharide-1-phosphate polymer are presented for the first time.
Keywords: Bacillus subtilis ; cell wall; teichoic acid; disaccharide-1-phosphate polymer; anionic polysaccharides; NMR spectroscopy

New isoform HvNHX3 of vacuolar Na+/H+-antiporter in barley: Expression and immunolocalization by T. V. Roslyakova; E. M. Lazareva; N. V. Kononenko; A. V. Babakov (549-556).
The gene HvNHX3 encoding a new isoform of vacuolar Na+/H+-antiporter was identified in barley. This gene is expressed in roots and leaves of barley seedlings, and it encodes a protein consisting of 541 amino acid residues with pre-dicted molecular weight 59.7 kDa. It was found that by its amino acid sequence HvNHX3 is closest to the Na+/H+-antiporter HbNHX1 of wild type from Hordeum brevisibulatum that grows on salt-marsh (solonchak) soils (95% homology). The expression of HvNHX3 during salt stress is increased several-fold in roots and leaves of barley seedlings. At the same time, the amount of HvNHX3 protein in roots does not change, but in leaves it increases significantly. It was shown using HvNHX3 immunolocalization in roots that this protein is present in all tissues, but in control plants it was clustered and in experimental plants after salt stress it was visualized as small granules. It has been proposed that HvNHX3 is converted into active form during declusterization. The conversion of HvNHX3 into its active form along with its quantitative increase in leaves during salt stress activates Na+/H+-exchange across the vacuolar membrane and Na+ release from cytoplasm, and, as a consequence, an increase of salt stress tolerance.
Keywords: barley; tonoplast; Na+/H+-antiporter; cDNA; immunolocalization; salt stress

Bacterial NAD-dependent Taq and Tth DNA ligases are capable of significantly increasing the yield of long PCR products when the amplification is carried out using bacterial family A DNA polymerases, e.g. Taq or Tth DNA polymerases, or with enzymatic blends containing these polymerases. We also show that Taq and Tth DNA ligases improve the results of PCR in the absence of NAD and therefore in the absence of DNA ligase activity. These observations suggest that bacterial DNA ligases can interact with these DNA polymerases, presumably as accessory proteins, thereby enhancing the efficiency of DNA polymerization.
Keywords: long PCR; Taq DNA polymerase; Taq DNA ligase

Glycogen phosphorylase b and phosphorylase kinase binding to glycogen under molecular crowding conditions. Inhibitory effect of FAD by N. A. Chebotareva; A. V. Meremyanin; V. F. Makeeva; T. B. Eronina; B. I. Kurganov (562-568).
Dynamic light scattering was used to study the interaction of phosphorylase kinase (PhK) and glycogen phos-phorylase b (Phb) from rabbit skeletal muscle with glycogen under molecular crowding conditions arising from the presence of 1 M trimethylamine N-oxide and at physiological ionic strength. The mean value of hydrodynamic radius of the initial glycogen particles was 52 nm. Crowding stimulated Phb and PhK combined binding on glycogen particles. Two-stage character of PhK binding to glycogen particles containing adsorbed Phb was found in the presence of the crowding agent. At the initial stage, limited size particles with hydrodynamic radius of ∼220 nm are formed, whereas the second stage is accompanied by linear growth of hydrodynamic radius. Flavin adenine dinucleotide (FAD) selectively inhibited PhK binding at the second stage. The data indicate that in the first stage Phb is involved in PhK binding by glycogen particles containing adsorbed Phb, whereas PhK binding in the second stage does not involve Phb.
Keywords: phosphorylase kinase; glycogen phosphorylase b ; crowding; trimethylamine N-oxide; glycogen; FAD; dynamic light scattering

Isolation and properties of fungal β-glucosidases by O. G. Korotkova; M. V. Semenova; V. V. Morozova; I. N. Zorov; L. M. Sokolova; T. M. Bubnova; O. N. Okunev; A. P. Sinitsyn (569-577).
Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).
Keywords: β-glucosidase; Aspergillus japonicus ; Penicillium verruculosum ; Trichoderma reesei ; saccharification; transglucosidation; HPLC

It was found that in medium with low ionic strength nucleoside diphosphate kinase (NDPK) solubilization from the outer membrane of liver mitochondria could be partially reversed by the addition of 3.3 mM MgCl2. Complete rebinding of the enzyme after the addition of MgCl2 was observed when the mitochondrial washing and storage medium contained leupeptin, an inhibitor of cathepsins. It was demonstrated that leupeptin and another inhibitor of cysteine proteinases, E-64, do not influence the rate of NDPK solubilization as well as its solubilized and membrane-associated activity. We conclude that NDPK becomes sensitive to proteolysis only after its solubilization; proteolysis does not affect the part of the enzyme molecule that is responsible for catalysis. After solubilization of NDPK in the absence of leupeptin, cathepsins damage sites of its binding on the membranes. The rate of the enzyme solubilization is dependent on the pH of the storage medium (pH 6.0–8.0); it decreases with increase in pH. It was shown that in the medium with high ionic strength, MgCl2 does not reverse pH-dependent NDPK solubilization, but solubilization could be reversed by increase in medium pH in the presence of E-64 and BSA. The physiological importance of these results is discussed.
Keywords: nucleoside diphosphate kinase; mitochondria; outer compartment; solubilization; liver