Biochemistry (Moscow) (v.75, #1)

In this review, we have compiled the data on pharmacological activities associated with endogenous purine release related enzymes—nucleases (DNases, RNases, and phosphodiesterases). The results of studies on toxic effects of these enzymes, emphasizing the future directions in this field, are summarized. One of the major problems facing toxicologists is the identification and characterization of specific venom nucleases since they share similar substrate specificities and biochemical properties. In this review, we have attempted to clarify some of the discrepancies about these enzymes. Further, we have tried to correlate the existence of nuclease enzymes in relation to endogenous release of purines, a multitoxin, during snake envenomation, and we also discuss the possible actions of purines. We hope that this review will stimulate renewed interest among toxicologists to biologically characterize these enzymes and elucidate their role in envenomation.
Keywords: purines; DNase; RNase; phosphodiesterase; snake envenomation; adenosine

Here we discuss some common mechanisms of microtubule-dependent active transport of nonmembranous components in animal cells. We summarize data about mRNA, cytoskeletal elements, structural proteins, and signaling complexes transport. We also characterize the series of molecular interactions that connect nonmembranous cargoes and microtubules and describe the regulatory pathways for these interactions.
Keywords: cytoskeleton; microtubules; kinesin; dynein; transport; mRNA; signal transduction

On mild acid degradation of the lipopolysaccharide of Escherichia coli O108, the O-polysaccharide was isolated and studied by sugar analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy. The polysaccharide was found to contain an unusual higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). The following structure of the tetrasaccharide repeating unit of the polysac-charide was established: →4)-α-8eLegp5Ac7Ac-(2→6)-α-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-α-D-GlcpNAc-(1→. Functions of the E. coli O108 antigen biosynthetic genes, including seven putative genes for synthesis of 8eLeg5Ac7Ac, were assigned by sequencing the O-antigen gene cluster along with comparison with gene databases and known biosynthetic pathways for related nonulosonic acids.
Keywords: Escherichia coli ; lipopolysaccharide; O-antigen; biosynthesis; O-polysaccharide structure; O-antigen gene cluster; nonulosonic acid

Femtosecond formation dynamics of primary photoproducts of visual pigment rhodopsin by O. A. Smitienko; M. N. Mozgovaya; I. V. Shelaev; F. E. Gostev; T. B. Feldman; V. A. Nadtochenko; O. M. Sarkisov; M. A. Ostrovsky (25-35).
The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400–720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin570 with vibrationally-excited all-trans-retinal and rhodopsin498 in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm−1 and 44, 142 cm−1 for photorhodopsin570 and for rhodopsin498, respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.
Keywords: rhodopsin; photorhodopsin; bathorhodopsin; femtosecond time-resolved laser absorption spectroscopy; coherent reaction; wave-packet

Protein trypsin inhibitor from potato tubers by T. A. Revina; G. V. Kladnitskaya; N. G. Gerasimova; E. L. Gvozdeva; T. A. Valueva (36-40).
A protein of 22 kDa designated as PKTI-22 was isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and purified to homogeneity using CM-Sepharose CL-6B ion-exchange chromatography. The protein efficiently suppressed the activity of trypsin, affected chymotrypsin less, and did not affect subtilisin Carlsberg. The N-terminal sequence of PKTI-22 (20 amino acid residues) was found to be highly homologous with the amino acid sequences of the potato Kunitz-type proteinase inhibitors of group B (PKPI-B) that were aligned from the corresponding gene sequences and was identical to the sequence (from the 2nd to the 20th residue) of the recombinant protein PKPI-B10. These data together with the observed similarity of the properties of two proteins indicate that the PKTI-22 protein is encoded by the PKPI-B10 gene.
Keywords: Kunitz-type proteinase inhibitors; trypsin; potato tubers

Isolation and properties of xyloglucanases of Penicillium sp. by O. A. Sinitsyna; E. A. Fedorova; A. G. Pravilnikov; A. M. Rozhkova; A. A. Skomarovsky; V. Yu. Matys; T. M. Bubnova; O. N. Okunev; Yu. P. Vinetsky; A. P. Sinitsyn (41-49).
Using chromatographic technique, xyloglucanase (XG) A (25 kDa, pI 3.5, 12th glycosyl hydrolase family) was isolated from the enzyme complex secreted by the mycelial fungus Penicillium canescens, and xyloglucanases XG 25 (25 kDa, pI 4.1, 12th glycosyl hydrolase family) and XG 70 (70 kDa, pI 3.5, 74th glycosyl hydrolase family) were isolated from the enzyme complex of Penicillium verruculosum. Properties of the isolated enzymes (substrate specificity, optimal ranges of pH and temperature for enzyme activity and stability, effect of metal ions on catalytic activity) were compared with the properties of xyloglucanases XG 32 of Aspergillus japonicus, XG 78 of Chrysosporium lucknowense, and XG of Trichoderma reesei. The gene xegA encoding XG A of P. canescens was isolated, and the amino acid sequence of the corresponding protein was determined.
Keywords: xyloglucanase; xyloglucan; Penicillium canescens ; Penicillium verruculosum

In this work, high ΔμH+-dependent succinate oxidase activity has been demonstrated for the first time with membrane vesicles isolated from Bacillus subtilis. The maximal specific rate of succinate oxidation by coupled inside-out membrane vesicles isolated from a B. subtilis strain overproducing succinate:menaquinone oxidoreductase approaches the specific rate observed with the intact cells. Deenergization of the membrane vesicles with ionophores or alamethicin brings about an almost complete inhibition of succinate oxidation. An apparent K m for succinate during the energy-dependent succinate oxidase activity of the vesicles (2.2 mM) is higher by an order of magnitude than the K m value measured for the energy-independent reduction of 2,6-dichlorophenol indophenol. The data reveal critical importance of ΔμH+ for maintaining active electron transfer by succinate:menaquinone oxidoreductase. The role of ΔμH+ might consist in providing energy for thermodynamically unfavorable menaquinone reduction by succinate by virtue of transmembrane electron transport within the enzyme down the electric field; alternatively, ΔμH+ could play a regulatory role by maintaining the electroneutrally operating enzyme in a catalytically active conformation.
Keywords: succinate:menaquinone oxidoreductase; ΔμH+ ; Bacillus subtilis ; electrogenesis; menaquinone; fumarate

Substrate specificity and biochemical properties of M3.BstF5I DNA methyltransferase from the BstF5I restriction-modification system by V. A. Chernukhin; V. V. Kuznetsov; D. A. Gonchar; Yu. G. Kashirina; N. A. Netesova; S. Kh. Degtyarev (63-71).
Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of λ phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min−1) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5′-GGATC-3′ DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5′-GGATG-3′ recognition site.
Keywords: DNA methyltransferase; enzyme kinetics; site specificity

Biochemical and morphological changes in dormant (“Nonculturable”) Mycobacterium smegmatis cells by E. G. Salina; Yu. A. Zhogina; M. O. Shleeva; G. M. Sorokoumova; A. A. Selishcheva; A. S. Kaprelyants (72-80).
Biochemical and morphological changes have been studied during transition of Mycobacterium smegmatis cells into their dormant (“nonculturable”) state. A significant fraction of the population of irreversibly “nonculturable” (NC) cells has a thicker cell wall, condensed cytoplasm, and a decreased number of ribosomes. The lipid contents in the NC cells are lower than in the metabolically active cells, with a relatively decreased amount of phospholipid and neutral lipid. Free mycolic acids, which are abundant in metabolically active cells, were not found in the NC cells. The NC forms are also characterized by decreased respiratory activity on endogenous substrates; however, the respiratory chain enzymes retain their activities in the isolated membranes. Activities of the Krebs cycle and glyoxylate cycle enzymes are markedly decreased. Despite a significant decrease in metabolic activity, NC cells possess membrane potential that seems to provide for reversibility of the NC state of mycobacteria, i.e. their capability of reactivating.
Keywords: dormancy; “nonculturable” (NC) cells; latent tuberculosis infection; Mycobacterium smegmatis ; M. tuberculosis

An analysis of amino acid sequences and 3D structures of chloroplast, mitochondrial, and bacterial F1-ATPases revealed that in their α- and β-chains there are short highly conserved segments linking in pairs the catalytic and noncatalytic sites. The analysis was based on the reported effect of directed mutagenesis of amino acids forming these segments on catalytic properties of the F1-ATPases. It is proposed that one of these segments is responsible for transduction of a conformation signal from the noncatalytic to catalytic site upon ADP-for-ATP substitution at the noncatalytic site. At the catalytic site, this signal changes position of the terminal amino acid residue with respect to the adenine part of the molecule and results in a lower tightness of MgADP binding and its dissociation followed by enzyme activation. Mutagenesis of amino acids comprised by the two other segments was shown to produce an effect on the rate of cooperative catalysis, whereas the rate of single-site catalysis remained unaffected. This suggests that these segments are responsible for the cooperative mode of enzyme functioning.
Keywords: coupling factor F1 ; F1-ATPase; ATP synthase; noncatalytic sites

Molecular cloning of a cotton phosphatase gene and its functional characterization by Shi-You Lu; Guang-Rong Zhao; Ai-Min Wu; M. A. Jenks; Shuping Zhang; Jin-Yuan Liu (85-94).
In this study, we report isolation of a phosphatase gene designated GhHL1 from cotton and its functional characterization. GhHL1 transcripts were detected in all cotton tissues examined. Southern blotting analysis indicated that it exists in multiple-copies. Biochemical analysis showed that GhHL1 was Mg2+-dependent and cation-sensitive. Purified recombinant GhHL1 protein dephosphorylated both 3′,5′-bisphosphate nucleotide and inositol 1,4-bisphosphate, demonstrating dual 3′,5′-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. Overexpression of GhHL1 complemented yeast hal2 mutant and enhanced yeast growth under elevated NaCl or LiCl, showing a role in salt tolerance associated with ionic stress response. Taken together, these results show that GhHL1 is a functional and good candidate gene, which might be used to improve salt tolerance in plants.
Keywords: cotton; phosphatase; salt tolerance; enzyme activity; functional characterization

Production of hydrogen peroxide and nitric oxide following introduction of nitrate and nitrite into wheat leaf apoplast by L. V. Viktorova; N. N. Maksyutova; T. V. Trifonova; V. V. Andrianov (95-100).
Infiltration of wheat (Triticum aestivum L.) seedling leaves with excess of nitrate, nitrite, or the NO donor sodium nitroprusside leads to increase both in content of hydroperoxide and activity of peroxidase and decrease in superoxide dismutase (SOD) activity in the leaf apoplast. Polymorphism of extracellular peroxidases and the presence of Cu/Zn-SOD have been shown in apoplast. Using an ESR assay, a considerable increase in the level of NO following infiltration of leaf tissues with nitrite has been demonstrated. These data suggest development of both oxidative and nitrosative stresses in leaves exposed to high levels of nitrate or nitrite. A possible interplay of NO and reactive oxygen species in plant cells is discussed.
Keywords: wheat; Triticum aestivum L.; apoplast; nitrate; nitrite; nitric oxide; hydrogen peroxide; antioxidant enzymes

Calcium signal-initiated early activation of NF-κB in neurons is a neuroprotective event in response to kainic acid-induced excitotoxicity by Shu-Yan Li; Wen-Ge Sun; Yu-Hong Jia; Guo-Sheng Wu; Guo-Shun An; Ju-Hua Ni; Hong-Ti Jia (101-110).
We demonstrate that activation of nuclear factor κB (NF-κB) in neurons is neuroprotective in response to kainic acid (KA)-induced excitotoxicity. Combination of Western blotting, immunocytochemistry, and electrophoresis mobility shift assay showed that KA exposure induced a fast but transient nuclear translocation of the NF-κB p65 subunit and increased DNA-binding activity of NF-κB in primary cultured cortical neurons. The transient NF-κB activity was associated with upregulation of antiapoptotic Bcl-xL and XIAP gene products revealed by real-time PCR. Knockdown of p65 decreased neuronal viability and antiapoptotic gene expression. In addition, we showed that KA-stimulated DNA-binding activity of NF-κB was associated with reactive oxygen species and calcium signals, using AMPA/KA receptor antagonist, calcium chelator, and antioxidant. These results suggest that the fast and transient activation of NF-κB initiated by calcium signals is one of the important proximal events in response to KA-induced excitotoxicity, which has neuroprotective effect against KA-induced apoptosis.
Keywords: NF-κB; kainic acid; ROS; calcium; AMPA/KA receptor; neuronal survival/death

The functional residues of z-class glutathione S-transferase were identified by screening inactive point mutants from a random mutagenesis library. First, a random mutant library was constructed using error-prone polymerase chain reaction, and then candidate inactive mutants were screened by a high-throughput colorimetric assay. Twenty-five mutants were obtained, and 12 that formed inclusion bodies were discarded. The remaining 13 mutants that expressed soluble protein were used for accurate quantification of enzymatic activity and sequencing. The mutants W15R, C19Y, R22H/K83E, P61S, S73P, S109P, and Q112R were found to have activity lower than 1% of the wild-type and were considered as “inactive mutants”, whereas the mutants K83E, Q102R, and L147F still have a large fraction of the activity and were thus considered as “partially inactivated mutants”. Molecular modeling experiments disclosed that mutations resulting in inactivation of the enzyme were found in or near the binding pocket, whereas mutations resulting in partial inactivation were distant from both substrates. The role of the residue Ser73 in the enzyme was verified by site-directed mutagenesis. The result suggested that screening inactive point mutants from a random mutagenesis library is an efficient way of identifying functional residues in enzymes.
Keywords: functional residues; random mutation; site-directed mutagenesis; Arabidopsis thaliana ; z-class glutathione S-transferase

YB-1 is capable of forming extended nanofibrils by O. M. Selivanova; S. G. Guryanov; G. A. Enin; M. A. Skabkin; L. P. Ovchinnikov; I. N. Serdyuk (115-120).
Here we are the first to report that multifunctional Y-box binding protein 1 (YB-1) forms extended fibrils with a diameter of 15–20 nm. The YB-1 fibrils were visualized by atomic force and electron microscopy after 1-h incubation in solution with 2 M LiCl. Their length grew with incubation time and could exceed 10 μm; their shape is helical or zigzag-like. They possess polarity and tend to associate with one another to give structures of a higher order, like ribbons or bundles. The YB-1 fibrillar architecture has a distinct periodicity with a repeat unit of about 52 nm.
Keywords: YB-1; protein fibrils; atomic force microscopy; electron microscopy; high ionic strength

Israel Moiseevich Gelfand by V. P. Skulachev (121-121).