Biochemistry (Moscow) (v.73, #2)

Sphingolipid receptors by E. V. Dyatlovitskaya (119-122).
The role of sphingolipids as receptors of bacteria, viruses, and toxins and also as ligands of proteinaceous receptors involved in the cell-cell signaling in animals is considered.
Keywords: sphingosine-1-phosphate; sphingosine-1-phosphocholine; receptors; bacteria; viruses; toxins

Each of two hydrophobic subunits of Na+-translocating NADH:quinone oxidoreductase (NQR), NqrD and NqrE, contain a pair of strictly conserved cysteine residues within their transmembrane α-helices. Site-directed mutagenesis showed that substitutions of these residues in NQR of Vibrio harveyi blocked the Na+-dependent and 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive quinone reductase activity of the enzyme. However, these mutations did not affect the interaction of NQR with NADH and menadione. It was demonstrated that these conserved cysteine residues are necessary for the correct folding and/or the stability of the NQR complex. Mass and EPR spectroscopy showed that NQR from V. harveyi bears only a 2Fe-2S cluster as a metal-containing prosthetic group.
Keywords: Na+-translocating NADH:quinone oxidoreductase; sodium potential; Vibrio ; respiratory chain

Light damaging action of all-trans-retinal and its derivatives on rhodopsin molecules in the photoreceptor membrane by M. Yu. Loginova; Ye. V. Rostovtseva; T. B. Feldman; M. A. Ostrovsky (130-138).
We have reproduced the model system containing A2-rhodopsin, NR-PE, A2-PE, and ATR-dimer-PE in order to study photosensitized damage of rhodopsin within photoreceptor membranes of rod outer segments. We have demonstrated that irradiation of such a system with visible light (400–700 nm) distorts the most important functional property of native visual pigment—its ability to regenerate after addition of 11-cis-retinal in the dark. We have also shown that all-trans-retinal bound to membrane phospholipids and rhodopsin has less photosensitizing activity that free all-trans-retinal.
Keywords: visual pigment rhodopsin; all-trans-retinal; photosensitizers; photodamage

Influence of cultivation conditions on spatial structure and functional activity of OmpF-like porin from outer membrane of Yersinia pseudotuberculosis by O. D. Novikova; T. I. Vakorina; V. A. Khomenko; G. N. Likhatskaya; N. Yu. Kim; V. I. Emelyanenko; S. M. Kuznetsova; T. F. Solov’eva (139-148).
The influence of cultivation conditions of pseudotuberculosis bacteria on the spatial structure and the functional activity of nonspecific OmpF-like porin was studied by means of optical spectroscopy, scanning microcalorimetry, and bilayer lipid membrane technique. With this goal, porin samples isolated from microbial masses grown at different temperatures, nutrient medium densities, and growth phases were characterized. According to CD data, the porin samples under investigation represent β-sheet proteins. It was found that the protein isolated from the colonial culture of pseudotuberculosis bacteria grown at low temperature has the most compact structure. Using intrinsic protein fluorescence, it was shown that different conditions of pseudotuberculosis bacteria cultivation (temperature, medium, growth phase) led to the changes in spectral properties of porin fluorescence due to the redistribution of the contributions of tyrosine and different classes of tryptophan residues to the total protein emission. Heat inactivation of porin samples was studied using CD spectroscopy, intrinsic protein fluorescence, and scanning microcalorimetry. Spatial features of the porin samples were found to affect their functional activities. Considering all these data, it is possible to correlate the spatial structure and functional activity of porin samples isolated under different cultivation conditions of bacteria and the composition of the outer membrane lipid matrix.
Keywords: nonspecific OmpF-like porins; spatial structure; thermostability; functional activity; lipid matrix

Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons by N. K. Isaev; E. V. Stelmashook; U. Dirnagl; E. Yu. Plotnikov; E. A. Kuvshinova; D. B. Zorov (149-155).
Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 μM) and NBQX (10 μM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase—oligomycin, an uncoupler of oxidative phosphorylation—CCCP, a chelator of intracellular Ca2+-BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport—ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+]i. Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.
Keywords: hypoglycemia; reactive oxygen species; free radicals; mitochondria; calcium; cerebellar granule neuron; cell culture

5S rRNA-recognition module of CTC family proteins and its evolution by A. V. Korobeinikova; G. M. Gongadze; A. P. Korepanov; B. D. Eliseev; M. V. Bazhenova; M. B. Garber (156-163).
The effects of amino acid replacements in the RNA-binding sites of homologous ribosomal proteins TL5 and L25 (members of the CTC family) on ability of these proteins to form stable complexes with ribosomal 5S RNA were studied. It was shown that even three simultaneous replacements of non-conserved amino acid residues by alanine in the RNA-binding site of TL5 did not result in noticeable decrease in stability of the TL5-5S rRNA complex. However, any replacement among five conserved residues in the RNA-binding site of TL5, as well as of L25 resulted in serious destabilization or complete impossibility of complex formation. These five residues form an RNA-recognition module in TL5 and L25. These residues are strictly conserved in proteins of the CTC family. However, there are several cases of natural replacements of these residues in TL5 and L25 homologs in Bacilli and Cyanobacteria, which are accompanied by certain changes in the CTC-binding site of 5S rRNAs of the corresponding organisms. CTC proteins and specific fragments of 5S rRNA of Enterococcus faecalis and Nostoc sp. were isolated, and their ability to form specific complexes was tested. It was found that these proteins formed specific complexes only with 5S rRNA of the same organism. This is an example of coevolution of the structures of two interacting macromolecules.
Keywords: bacterial 5S rRNA-binding proteins; CTC family proteins; ribosome

Isolation and oligomeric composition of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens by T. V. Tikhonova; E. S. Slutskaya; A. A. Filimonenkov; K. M. Boyko; S. Yu. Kleimenov; P. V. Konarev; K. M. Polyakov; D. I. Svergun; A. A. Trofimov; V. G. Khomenkov; R. A. Zvyagilskaya; V. O. Popov (164-170).
A new procedure for isolation of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens increasing significantly the yield of the purified enzyme is presented. The enzyme is isolated from the soluble fraction of the cell extract as a hexamer, as shown by gel filtration chromatography and small angle X-ray scattering analysis. Thermostability of the hexameric form of the nitrite reductase is characterized in terms of thermoinactivation and thermodenaturation.
Keywords: cytochrome c nitrite reductase; isolation; oligomeric composition; thermostability; thermodenaturation

Association of dinitrogenase reductase-activating glycohydrolase (DRAG) with membrane proteins of chromatophores has been investigated. The formation of a multicomponent complex between DRAG and membrane proteins was demonstrated in the presence of glutaraldehyde and EDC/NHS (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid sodium salt). Complex formation was observed both in native chromatophore membrane and in chromatophores treated with 0.5 M NaCl in the presence of homogeneous DRAG and glutaraldehyde in cross-reaction. The molecular weight of the complex was around 200 kD, which is consistent with the association of DRAG with three or more chromatophore membrane proteins. A specific complex with molecular weight of about 75 kD was formed only in the presence of EDC/NHS in the cross-linking reaction. It was demonstrated that ammonium transport protein and P11 protein are possible candidates for association with DRAG in chromatophore membranes.
Keywords: nitrogen fixation; nitrogenase; dinitrogenase reductase-activating glycohydrolase; cross-linking complex; membranes

Study of the interaction between rabbit cardiac contractile and regulatory proteins. An in vitro motility assay by L. V. Nikitina; G. V. Kopylova; D. V. Shchepkin; L. B. Katsnelson (178-184).
A series of experiments was performed in an in vitro motility assay with reconstructed thin filaments to obtain pCa-force relationships for cardiac isomyosins V1 and V3. Two concentrations of each isomyosin (200 and 300 μg/ml) on the surface of a flow cell were tested. Isometric force was estimated as the amount of actin-binding protein, α-actinin, stopping thin filament movement. It was found that the amount of α-actinin stopping the movement at saturating calcium concentration for V3 was twice higher than for V1 at both concentrations of isoforms. Hill coefficients of cooperativity (h) were determined for pCa-force relationships. The value of h did not differ significantly for isoforms at 300 μg/ml of protein (h was 1.56 for V1 and 1.54 for V3). However, the Hill coefficient was higher for V3 isoform at 200 μg/ml (h = 2.00 and 1.76 for V3 and V1, respectively). Importantly, the Hill coefficient increased for both isoenzymes when their concentrations were decreased. The connection between Hill coefficient and cooperative interactions between cardiac contractile and regulatory proteins is analyzed in detail.
Keywords: cardiac isomyosins; cooperativity between contractile and regulatory proteins

Investigation of glyceraldehyde-3-phosphate dehydrogenase from human sperms by Yu. Yu. Shchutskaya; Yu. L. Elkina; M. L. Kuravsky; E. E. Bragina; E. V. Schmalhausen (185-191).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDs) was purified from human sperms and properties of the enzyme were investigated. After sonication of sperms, the most part of GAPDs is associated with the insoluble cell fraction. Trypsin treatment results in the cleavage of part of the N-terminal domain of the enzyme yielding a soluble fragment that was purified by hydrophobic chromatography on Phenyl-Sepharose. The isolated fragment was shown to be a tetramer with molecular weight of approximately 150 kD (according to Blue Native PAGE) and composed of subunits of 40 kD (according to SDS-PAGE). The specific activity of the isolated fragment reached 374 U/mg. It is supposed that GAPDs exists in sperms as the tetrameric molecule bound to the fibrous sheath of the flagellum through the N-terminus of one or two subunits. Comparative study of the amino acid sequences of mammalian GAPDs revealed conservative cysteine residues (C21, C94, and C150) that are specific for the sperm isoenzyme and absent in the somatic isoenzyme. Residue C21 can be involved in the formation of the disulfide bond between the N-terminal domain of GAPDs and fibrous sheath proteins.
Keywords: glyceraldehyde-3-phosphate dehydrogenase; spermatozoa; fibrous sheath; trypsin

Structural diversity and endotoxic activity of the lipopolysaccharide of Yersinia pestis by S. V. Dentovskaya; I. V. Bakhteeva; G. M. Titareva; R. Z. Shaikhutdinova; A. N. Kondakova; O. V. Bystrova; B. Lindner; Y. A. Knirel; A. P. Anisimov (192-199).
The endotoxic activity of the lipopolysaccharides (LPS) with defined chemical structure from Yersinia pestis strains of various subspecies differing in their epidemic potential was studied. The LPS of two strains of Y. pestis ssp. caucasica and ssp. altaica, whose structures have not been studied earlier, were analyzed by high-resolution mass spectrometry. In addition to reported structural changes, an increase in the degree of LPS phosphorylation was observed when strain I-2377 (ssp. altaica) was cultivated at an elevated temperature. A high tumor necrosis factor α (TNF-α)-inducing activity observed for LPS samples from Y. pestis cultures grown at 25°C correlated with an increased degree of lipid A acylation, particularly, with the presence of the hexaacyl form of lipid A, which was absent from the LPS when bacteria were cultivated at 37°C. No correlation was found between the lethal toxicity of the LPS in vivo and its ability to induce TNF-α production in vitro.
Keywords: lipopolysaccharide; lipid A; endotoxin; tumor necrosis factor; cytokine-inducing activity; Yersinia pestis

Human small heat shock protein with molecular mass 22 kD (HSP22, HspB8) contains two Ser residues (Ser24 and Ser57) in consensus sequence RXS and is effectively phosphorylated by cAMP-dependent protein kinase in vitro. Mutation S24D did not affect, whereas mutations S57D or S24,57D prevented phosphorylation of HSP22 by cAMP-dependent protein kinase thus indicating that Ser57 is the primary site of phosphorylation. Phosphorylation (or mutation) of Ser57 (or Ser24 and Ser57) resulted in changes of the local environment of tryptophan residues and increased HSP22 sus-ceptibility to chymotrypsinolysis. Mutations mimicking phosphorylation decreased dissociation of HSP22 oligomer at low concentration without affecting its quaternary structure at high protein concentration. Mutations S24D, S57D, and especially S24,57D were accompanied by decrease of chaperone-like activity of HSP22 if insulin and rhodanase were used as substrates. Thus, phosphorylation by cAMP-dependent protein kinase affects the structure and decreases chaperone-like activity of HSP22 in vitro.
Keywords: small heat shock proteins; phosphorylation; structure; chaperone-like activity

Inhibition of tumor specific angiogenesis by amentoflavone by C. Guruvayoorappan; G. Kuttan (209-218).
The formation of new capillaries from existing blood vessels is critical for tumor growth and metastasis. In this study we report that amentoflavone, a biflavonoid from Biophytum sensitivum, could inhibit the process of angiogenesis. Amentoflavone at nontoxic concentrations (0.05–0.2 μg/ml) showed significant inhibition in the proliferation, migration, and tube formation of endothelial cells, which are key events in the process of angiogenesis. In vivo studies in C57BL/6 mice using amentoflavone showed remarkable inhibition (52.9%) of tumor directed capillary formation. Amentoflavone showed inhibitory effect on the production of various endogenous factors such as IL-1β, IL-6, TNF-α, GM-CSF, and VEGF that control the process of angiogenesis. Amentoflavone treatment could increase the production of IL-2 and TIMP-1, which could successfully shift the equilibrium towards an angiostatic condition. The antiangiogenic activity of amentoflavone was supported by its remarkable suppression in sprouting of microvessels from rat aorta. Our results also show that amentoflavone could inhibit the production of VEGF mRNA in B16–F10 cells. These findings indicate that amentoflavone inhibits angiogenesis by disrupting the integrity of endothelial cells and by altering the endogenous factors that are required for the process of neovascularization.
Keywords: amentoflavone; angiogenesis; endothelial cells; proinflammatory cytokines; tube formation

Molecular-genetic systems of development: Functional dynamics and molecular evolution by K. V. Gunbin; V. V. Suslov; N. A. Kolchanov (219-230).
This work for the first time compares results obtained with studies of parametric robustness of the Hh-and Dpp/BMP signal cascades responsible for morphogenesis and molecular evolution of the Hh-and Dpp/BMP cascade genes. There is a link between adaptive evolution of genes and those changes in kinetic parameters of the signal cascade models, which are critical for normal functioning of these cascades. Special attention is paid to events of the molecular evolution of the Hh-and Dpp/BMP cascade genes that matched with the emergence of the major taxonomic types and classes of Bilateria.
Keywords: Hh-and Dpp/BMP signal cascades; mathematical modeling; positive selection; Bilateria; compensatory changes

Interfacial atom pair analysis by Yong-Chao Li; Zong-Hao Zeng (231-233).
The relations of the binding free energies in a dataset of 69 protein complexes with the numbers of interfacial atom pairs, as well as with the atomic distances of the pairs, are analyzed. It is found that the interfacial main-chain atom pairs contribute more to the correlation than the interfacial side chain atom pairs do, and the polar atom pairs contribute more than the non-polar atom pairs do. Interfacial atom pairs with atomic distance in the range of 6–12 Å are the most important to explain the differences in binding free energies in the datasets.
Keywords: interfacial atom pairs; statistical potentials; complementarity