Biochemistry (Moscow) (v.73, #3)

Role of A-chain in functioning of the active site of human α-thrombin by M. V. Kolodzeiskaya; L. I. Sokolovskaya; G. L. Volkov (237-244).
This review summarizes current data suggesting that A-chain of the human α-thrombin molecule plays a role of allosteric effector in catalytic reactions with various substrates. Special attention is paid to the relationship between A-chain structure and catalytic activity of thrombin. The existence of this relationship is based on studies of natural mutation of A-chain of the α-thrombin molecule. Use of molecular and essential dynamics confirmed the role of A-chain in changes of conformation and catalytic properties of this enzyme; these changes involve residues located in the specificity sites and some inserting loops. Current knowledge on structure and properties of thrombin can be used for the development of new antithrombin agents.
Keywords: thrombin; allosterics; mutagenesis; protein C; modulation; thrombomodulin; molecular dynamics

A new Mg(II) complex of MgL(NO3)2 (here L = N1,N8-bis(1-methyl-4-nitropyrrole-2-carbonyl)triethylenetetramine) has been synthesized and characterized. The interactions between the Mg(II) complex and calf thymus DNA has been investigated using UV spectra, fluorescent spectra, viscosity, thermal denaturation, and molecular modeling. The cleavage reaction on plasmid DNA has been monitored by agarose gel electrophoresis. The experimental results show that the mode of binding of the complex to DNA is non-classical electrostatic action and the complex can cleave pBR322 DNA.
Keywords: DNA binding; N1,N8-bis(1-methyl-4-nitropyrrole-2-carbonyl)triethylenetetramine; Mg(II) complex; calf thymus DNA

Interaction between kringle and growth-factor-like domains in the urokinase molecule: Possible role in stimulation of chemotaxis by V. V. Stepanova; I. B. Beloglazova; Y. G. Gursky; R. S. Bibilashvily; Y. V. Parfyonova; V. A. Tkachuk (252-260).
The results presented in this paper suggest the presence of an interaction between the kringle-and the growth-factor-like urokinase domains. This interaction regulates chemotactic properties of urokinase. We also show that interaction of urokinase with its “classical” receptor (uPAR) has a “permissive” effect on the interactions between the kringle domain and other targets on the cell surface. On the basis of our data we can suggest that uPAR serves as an “adaptor” for urokinase, and the binding of urokinase kringle domain to its receptor causes immediate activation of intracellular signaling and induction of cell migration.
Keywords: urokinase; kringle domain; urokinase receptor; intermolecular interactions; cellular migration

Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and DNA polymerase β with various DNA structures imitating intermediates of DNA repair and replication were investigated by gel retardation and photoaffinity labeling. Photoaffinity labeling of APE1 and DNA polymerase β was accomplished by DNA containing photoreactive group at the 3′-end in mouse embryonic fibroblast (MEF) cell extract or for purified proteins. On the whole, modification efficiency was the same for MEF-extract proteins and for purified APE1 and DNA polymerase β depending on the nature of the 5′-group of a nick/gap in the DNA substrate. Some of DNA duplexes used in this work can be considered as short-patch (DNA with the 5′-phosphate group in the nick/gap) or long-patch (DNA containing 5′-sugar phosphate or 5′-flap) base excision repair (BER) intermediates. Other DNA duplexes (3′-recessed DNA and DNA with the 5′-hydroxyl group in the nick/gap) have no relation to intermediates forming in the course of BER. As shown by both methods, APE1 binds with the highest efficiency to DNA substrate containing 5′-sugar phosphate group in the nick/gap, whereas DNA polymerase β binds to DNA duplex with a mononucleotide gap flanked by the 5′-p group. When APE1 and DNA polymerase β are both present, a ternary complex APE1-DNA polymerase β-DNA is formed with the highest efficiency with DNA product of APE1 endonuclease activity and with DNA containing 5′-flap or mononucleotide-gapped DNA with 5′-p group. It was found that APE1 stimulates DNA synthesis catalyzed by DNA polymerase β, and a human X-ray repair cross-complementing group 1 protein (XRCC1) stimulates APE1 3′–5′ exonuclease activity on 3′-recessed DNA duplex.
Keywords: APE1; base excision repair; photoaffinity labeling

A full-length cDNA encoding ribosome-inactivating/antiviral protein from the leaves of Bougainvillea xbuttiana was recently isolated. The coding region of cDNA was cloned and expressed in Escherichia coli, and the protein product was designated as BBAP1 (Bougainvillea xbuttiana antiviral protein 1). BBAP1 showed ribonuclease activity against Torula yeast RNA. It also exhibited depurination activity against supercoiled pBlueScript SK+ plasmid DNA in a concentration dependent manner, and was found to convert nicked circular DNA into linear form only at higher concentration. On bioassay, BBAP1 exhibited antiviral activity against sunnhemp rosette virus infecting Cyamopsis tetragonoloba leaves in which 95% inhibition of local lesion formation was observed.
Keywords: Bougainvillea xbuttiana ; antiviral protein; BBAP1; RNase activity; DNase activity; antiviral activity

Membrane localization of the MAK-V protein kinase by S. V. Kalinichenko; E. V. Korobko; I. V. Korobko (278-282).
Activities of many proteins including protein kinases are often regulated by their dynamic association with specific intracellular compartments. MAK-V is an AMPK-like protein kinase with poorly characterized functions and mechanisms of action. Similarly to many other protein kinases, association of MAK-V with specific intracellular compartments could be essential for its proper functions. In this work, we studied subcellular distribution of exogenously produced and endogenous MAK-V proteins in mammalian cells using biochemical cell fractioning aiming to supplement data on MAK-V intracellular localization studied by immunocytochemical methods. We found that a significant portion of MAK-V protein in mammalian cells is associated with membranes. Moreover, MAK-V expressed in yeast was also targeted to membrane, thus suggesting an evolutionarily conservative mechanism of MAK-V membrane association. Based on the ability of various MAK-V deletion mutants to localize to membrane and comparison of MAK-V amino acid sequences from different species, we suggest a possible mechanism governing MAK-V association with intracellular membranes.
Keywords: protein kinase MAK-V; membrane localization; ubiquitin-associated domain

Lipid rafts association of synaptotagmin I on synaptic vesicles by Ji-Hua Lv; Li He; Sen-Fang Sui (283-288).
We confirmed the raft association of synaptotagmin I (syt I) in synaptic vesicles by sucrose density gradient centrifugation, cholesterol depletion, and temperature dependence, and Ca2+ was found to positively regulate this association. Furthermore, using syt I mutants we found that the transmembrane domain (TMD) of syt I plays an important role in localizing syt I into the lipid rafts of synaptic vesicles, and the raft association of the TMD can be regulated by its phosphorylation status.
Keywords: lipid rafts; synaptic vesicles; synaptotagmin I

We have isolated and purified endogenous cytosolic tankyrase from human embryonic kidney cells of line 293. Our data confirm a model of De Rycker and Price who consider that tankyrase is a master scaffolding protein capable of regulating assembly of large protein complexes. We have also studied kinetic characteristics of tankyrase in the complex, pH dependence of the enzyme activity, and its physicochemical properties.
Keywords: tankyrase; poly(ADP-ribose); PARP; ADP-ribosylation; kidney

The effects of peroxisome proliferator activated receptors α and γ (PPAR-α and PPAR-γ) and retinoid X receptor (RXR) agonists upon synthesis and accumulation of lipids in murine C57B1 macrophages during inflammation induced by injection of zymosan and Escherichia coli lipopolysaccharide (LPS) have been studied. It is significant that intraperitoneal injection of zymosan (50 mg/kg) or LPS (0.1 mg/kg) in mice led to a dramatic increase of [14C]oleate incorporation into cholesteryl esters and triglycerides and [14C]acetate incorporation into cholesterol and fatty acids in peritoneal macrophages. Lipid synthesis reached its maximum rate 18–24 h after injection and was decreased 5–7 days later to control level after LPS injection or was still heightened after zymosan injection. In macrophages obtained in acute phase of inflammation (24 h), degradation of 125I-labeled native low density lipoprotein (NLDL) was 4-fold increased and degradation of 125I-labeled acetylated LDL (AcLDL) was 2–3-fold decreased. Addition of NLDL (50 μg/ml) or AcLDL (25 μg/ml) into the incubation medium of activated macrophages induced 9–14-and 1.25-fold increase of cholesteryl ester synthesis, respectively, compared with control. Addition of NLDL and AcLDL into the incubation medium completely inhibited cholesterol synthesis in control macrophages but had only slightly effect on cholesterol synthesis in activated macrophages. Injection of RXR, PPAR-α, or PPAR-γ agonists—9-cis-retinoic acid (5 mg/kg), bezafibrate (10 mg/kg), or rosiglitazone (10 mg/kg), respectively—30 min before zymosan or LPS injection led to significant decrease of lipid synthesis. Ten hour preincubation of activated in vivo macrophages with the abovementioned agonists (5 μM) decreased cholesteryl ester synthesis induced by NLDL and AcLDL addition into the cell cultivation medium. The data suggest that RXR, PPAR-α, or PPAR-γ agonists inhibited lipid synthesis and induction of cholesteryl ester synthesis in inflammatory macrophages caused by capture of native or modified LDL.
Keywords: macrophages; lipid metabolism; PPAR; RXR; inflammation

Administration of cortisol to an animal induces tyrosine aminotransferase (TAT) in the liver. A similar effect was observed after stimulation of resident liver macrophages (Kupffer cells) by dextran sulfate. Actinomycin D completely blocks enzyme induction both by cortisol and dextran sulfate, whereas their combined effect gives an additive result. In primary culture of hepatocytes, dextran sulfate inhibits TAT activity, but conditioned macrophage medium reliably increases enzyme activity in hepatocytes. However, incubation of isolated macrophages in the presence of dextran sulfate and such medium transfer into hepatocyte culture results in even more pronounced increase in TAT activity. In a combined culture of hepatocytes and non-parenchymal liver cells, reproducing intercellular interactions in vitro, cortisol and non-parenchymal cells exhibit an additive effect on TAT activity. These results show that liver macrophages release a factor of unknown nature launching the mechanism of TAT induction independently of cortisol, a classic TAT inducer.
Keywords: tyrosine aminotransferase; glucocorticoids; macrophage; hepatocyte; liver; intercellular interactions; dextran sulfate

Extracellular yeast-lytic enzyme of the bacterium Lysobacter sp. XL 1 by O. A. Stepnaya; I. M. Tsfasman; I. A. Chaika; T. A. Muranova; I. S. Kulaev (310-314).
An enzyme exhibiting yeast-lytic activity has been isolated from the culture liquid of the bacterium Lysobacter sp. XL. 1. The optimal conditions for the hydrolysis of Saccharomyces cerevisiae cells by the enzyme have been established: 0.15 M sodium acetate buffer, pH 6.0, 50°C. The yeast-lytic activity of the enzyme is inhibited by EDTA, p-chloromercuribenzoate, and phenylmethylsulfonyl fluoride. According to the data of SDS-PAGE, the molecular weight of the protein is 36 kD. The enzyme hydrolyzes casein, hemoglobin, and synthetic peptide Abz-Ala-Ala-Phe-pNA, i.e. it exhibits proteolytic activity. The properties of the enzyme and its molecular weight correspond to those of a previously isolated extracellular metalloproteinase. The N-terminal amino acid sequence of the protein exhibits 67% homology with the N-terminal sequence of achromolysine of Achromobacter lyticus (EC 3.4.24.-).
Keywords: metalloproteinase; lysoamidase; Lysobacter sp; yeast-lytic enzymes

Proteins and peptides of the salivary gland secretion of medicinal leeches Hirudo verbana, H. medicinalis, and H. orientalis by I. P. Baskova; E. S. Kostrjukova; M. A. Vlasova; O. V. Kharitonova; S. A. Levitskiy; L. L. Zavalova; S. A. Moshkovskii; V. N. Lazarev (315-320).
The protein and peptide composition of medicinal leech salivary gland secretion (SGS) was analyzed in preparations obtained in July from three species—Hirudo verbana, H. medicinalis, and H. orientalis. Two-dimensional electrophoresis (molecular mass 10–150 kD and pI 3–10) revealed no distinctions in the distribution of over 100 silver-stained proteins. Differences were noted only in intensity of 10 protein spots at 30–90 kD and pI 4.7–7.5. Mass spectrometric profiling of SGS of the three leech species using the Zip-Tip/golden chip scheme and cation-exchanging chips CM-10 revealed over 50 components in SGS of each of the three leech species. It was noted that 30–40% of the individual masses of the SGS of each leech species fall within the masses present in SGS of at least one of the two other species. This rather small part of the total mass may be indicative of a high polymorphism of amino acid sequences or a high frequency of posttranslational modifications of the SGS proteins and peptides. Calculation of Jacquard’s coefficient showed that H. medicinalis and H. orientalis are closest to each other in SGS composition, which is consistent with data in the literature on the phylogenetic relationship between these two species of medicinal leech. Comparison of detected molecular masses with those of six known biologically active compounds produced by medicinal leeches revealed their uneven distribution in SGS of each of the three medicinal leech species. This opens prospects for using certain species of medicinal leech for targeted therapy of various pathologies.
Keywords: proteomic analysis; two-dimensional electrophoresis; SELDI mass spectrometry; salivary gland secretion; medicinal leech; Hirudo verbana ; H. medicinalis ; H. orientalis

In the present study, we found that ionic interactions are not essential for the binding of nucleoside diphosphate kinase of liver mitochondria outer compartment to outer mitochondrial membrane and that the proportion of the enzyme activity involved in functional coupling with oxidative phosphorylation (we demonstrated the existence of functional coupling earlier) is only 17%. Additional evidence was obtained that functionally coupled activity of nucleoside diphosphate kinase is associated with the outer surface of mitochondria. Dextran (10%) did not increase functional coupling. The physological importance of these effects is discussed.
Keywords: nucleoside diphosphate kinase; mitochondria; outer compartment; localization; oxidative phosphorylation; functional coupling

Brain cathepsin B cleaves a caspase substrate by A. A. Yakovlev; A. Yu. Gorokhovatsky; M. V. Onufriev; I. P. Beletsky; N. V. Gulyaeva (332-336).
We show that an enzyme exists in rat brain capable of cleaving the caspase-3 specific peptide substrate Ac-DEVD-AMC at low pH. The enzyme shows properties of a cysteine protease and is localized, predominantly, in lysosomes. We have purified this enzyme from rat brain and identified it by MALDI-TOF MS. The enzyme possessing “acidic” DEVDase activity in rat brain appears to be cathepsin B. It remains obscure, whether cathepsin B participates in cleavage of caspase-3 substrates in vivo. We suggest that under certain conditions (e.g. in hypoxia) cathepsin B participates in cleavage of caspase-3 substrates in brain cells.
Keywords: caspase-3; cathepsin B; Ac-DEVD-AMC

Hyperactivation of succinate dehydrogenase in lymphocytes of newborn rats by N. V. Khunderyakova; M. V. Zakharchenko; A. V. Zakharchenko; M. N. Kondrashova (337-341).
We measured the activity of mitochondrial succinate dehydrogenase (SDH) within cells, in media with near-physiological composition, in lymphocytes immobilized in a blood smear on glass. SDH activity was studied in newborn rats characterized by natural hyperadrenergic status and also in adult animals injected with epinephrine. In most newborns very high activities were recorded, which exceeded the activities in adults at rest 7–8-fold or 3-fold according to the conventional calculation, or more than 30-and 6-fold according to our more precise calculation. The findings support our concept about a selective interaction between adrenergic stimulation and oxidation of succinic acid. According to this concept, epinephrine and norepinephrine specifically activate oxidation of succinic acid, whereas blood micromolar concentrations of the latter stimulate the release of catecholamines (the receptor-mediated signaling effect). This interaction is half of a substrate-hormonal regulatory system responsible for connection of vegetative nervous system with oxidation in mitochondria of the innervated organs. The increase in succinate oxidation by catecholamines includes activation of the faster pathways of succinate generation than the complete Krebs cycle, in particular, the glyoxylate cycle that is shown in the newborn rats in the present study.
Keywords: newborns; epinephrine; succinate dehydrogenase; succinic acid; glyoxylate cycle

siRNAs targeting mouse myostatin by V. A. Furalyov; I. V. Kravchenko; V. P. Khotchenkov; V. O. Popov (342-345).
Eight different mouse myostatin small interfering RNA (siRNAs) were synthesized and tested. Five siRNAs showed a pronounced biological effect reducing myostatin mRNA content. For two of them, the myostatin mRNA level was reduced 3-and 4-fold, respectively. The obtained siRNAs can be used for study of biological effects of myostatin, both in vitro and in vivo.
Keywords: myostatin; siRNA; RNA interference; myoblasts

Evolution of DNA polymerase ι structure and function in eukaryotes by A. V. Makarova; V. Z. Tarantul; L. V. Gening (346-352).
Analysis of DNA polymerase ι (Pol ι) enzymic activity in different classes of eukaryotes has shown that error-prone activity of this enzyme can be found only in mammals, and that it is completely absent from organisms that are at lower stages of development. It was supposed that the emergence of the error-prone Pol ι activity in mammals is caused by structural alteration of the active center. Possible functions of error-prone Pol ι in higher eukaryotes are discussed.
Keywords: DNA polymerase ι ; error-prone activity; hypermutagenesis

Human heart LIM (hhLIM), a muscle-specific expressed transcriptional coactivator of cardiac hypertrophy related gene, stimulates transcriptional activation of the ANF gene promoter in H9C2 cells. We revealed that the N-terminal half of hhLIM mediated this activation, in which the LIM domain 1 and protein kinase C phosphorylation site are important, especially the LIM domain 1. Further, mutagenesis of the conserved Cys in the LIM domain 1 abolished its ability to activate cardiac hypertrophy. These findings suggest that hhLIM is a typical LIM family member with powerful transcription activation.
Keywords: hhLIM; LIM domain; cardiac hypertrophy; ANF

Cytoskeleton inhibitors combined with TRAIL induce apoptosis in HeLa carcinoma cells overexpressing antiapoptotic protein Bcl-2 by M. E. Gasparian; L. V. Domnina; O. Yu. Ivanova; D. S. Izyumov; A. Yu. Lomakin; E. N. Popova; A. V. Yagolovich; O. Yu. Pletjushkina; D. A. Dolgikh; B. V. Chernyak (358-362).
TRAIL (Apo2L), a cytokine from the family of tumor necrosis factors (TNF), causes apoptosis in various types of tumor cells but is not toxic for normal cells. Recombinant TRAIL obtained using an original method stimulates the release of cytochrome c from mitochondria into the cytoplasm and apoptosis in HeLa carcinoma cells. Expression of oncoprotein Bcl-2 in these cells blocks both processes. The microtubule inhibitors taxol, nocodazole, and colcemid, as well as an inhibitor of actin microfilaments cytochalasin D, enhance the action of TRAIL and allow it to overcome protection caused by overexpression of Bcl-2. This effect is not associated with enhancement of early steps of TRAIL-dependent apoptosis leading to activation of caspase-8 and Bid protein. The inactivation of Bcl-2 also does not define the effect of cytoskeleton inhibitors. It is supposed that destruction of cytoskeleton alters the mechanism of the TRAIL-(or TNF)-dependent cytochrome c release from mitochondria by making it resistant to Bcl-2. The combined use of cytoskeleton inhibitors, which are antitumor drugs, with the recombinant TRAIL preparations may be efficient in therapy of tumors resistant to traditional chemotherapy.
Keywords: apoptosis; cytokines; mitochondria; cytoskeleton; Bcl-2

Notable and anniversary dates in biochemistry for 2008 by N. P. Voskresenskaya (363-365).