Biochemistry (Moscow) (v.74, #7)

Function of SIRT1 in physiology by Xing-Xing Kong; Rui Wang; Xiao-Jun Liu; Liu-Luan Zhu; Di Shao; Yong-Sheng Chang; Fu-De Fang (703-708).
Sirtuins were originally defined as a family of oxidized nicotinamide adenine nucleotide (NAD+)-dependent enzymes that deacetylate lysine residues on various proteins. The sirtuins are remarkably conserved throughout evolution from archae to eukaryotes. They were named after their homology to the Saccharomyces cerevisiae gene silent information regulator 2 (Sir2). The mammalian sirtuins, SIRT1-7, are implicated in a variety of cellular functions ranging from gene silencing, control of the cell cycle and apoptosis, and energy homeostasis. As SIRT1 is a nuclear protein and is the mammalian homolog most highly related to Sir2, it has been the focus of a large number of recent studies. Here we review some of the current data related to SIRT1 and discuss its mode of action and biological role in cellular and organismal models.
Keywords: SIRT1; metabolism; calorie restriction; apoptosis

Purification and biochemical characterization of a D-galactose binding lectin from Japanese sea hare (Aplysia kurodai) eggs by S. M. A. Kawsar; R. Matsumoto; Y. Fujii; H. Yasumitsu; C. Dogasaki; M. Hosono; K. Nitta; J. Hamako; T. Matsui; N. Kojima; Y. Ozeki (709-716).
A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80°C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, β-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-α- and methyl-β-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k ass) and dissociation rate constant (k diss) were determined for the lectin to be 4.3·105 M−1·sec−1 and 2.2·10−3 sec−1, respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.
Keywords: Aplysia kurodai ; cell proliferating inhibition; D-galactose-binding lectin; galacturonic acid; sea hare; surface plasmon resonance

Molecular characterization of tumor associated glyceraldehyde-3-phosphate dehydrogenase by S. Patra; S. Ghosh; S. Bera; A. Roy; S. Ray; M. Ray (717-727).
Here we describe the purification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from normal leukocytes of healthy subjects and leukocytes of chronic myeloid leukemia (CML) patients and from normal mouse muscle and sarcoma tissue. The data indicate that some properties of GAPDH of leukocytes of CML patients and sarcoma tissues are similar and also similar to those of EAC (Ehrlich ascites carcinoma) cellular GAPDH but distinctly different from those of the normal cellular GAPDH. Polyclonal antiserum raised against the 54 kDa subunit of EAC cell GAPDH strongly reacted with GAPDH of leukocytes of CML patients and sarcoma tissue GAPDH only and weakly reacted with GAPDH of normal leukocyte and normal muscle and a variety of other tissues of normal rats. Both the subunits of GAPDH of sarcoma tissues were partially sequenced from the N-terminus and compared with the known sequences of GAPDH. The altered properties of GAPDH of three different malignant sources might be common feature of all malignant cells, which is discussed in relation to glycolysis and malignant aberrations.
Keywords: glyceraldehyde-3-phosphate dehydrogenase; sarcoma; leukemia; cancer marker

Identification and mapping of ten new potential insulators in the FXYD5-COX7A1 region of human chromosome 19q13.12 by D. A. Didych; S. B. Akopov; E. V. Snezhkov; N. V. Skaptsova; L. G. Nikolaev; E. D. Sverdlov (728-733).
A positive-negative selection system revealed 10 potential insulators able to block enhancer interaction with promoter in the 106 bp human chromosome 19 region between genes FXYD5 and COX7A1. Relative positions of insulators and genes are in accord with the hypothesis that insulators subdivide genomic DNA into independently regulated loop domains.
Keywords: human genome; functional mapping; annotations of DNA sequences; insulator

Structural and kinetic features of family I inorganic pyrophosphatase from Vibrio cholerae by E. V. Rodina; V. R. Samygina; N. N. Vorobyeva; T. S. Sitnik; S. A. Kurilova; T. I. Nazarova (734-742).
In this paper, kinetic properties of a soluble inorganic pyrophosphatase of family I from Vibrio cholerae (V-PPase), intestinal pathogen and causative agent of human cholera, are characterized in detail, and the crystal structure of a metal-free enzyme is reported. Hydrolytic activity of V-PPase has been studied as a function of pH, concentration of metal cofactors (Mg2+ or Mn2+), and ionic strength. It has been found that, despite the high conservation of amino acid sequences for the known bacterial PPases of family I, V-PPase differs from the other enzymes of the same family in a number of parameters. Dissociation constants of V-PPase complexed with Mg2+ or Mn2+ were essentially the same as for Escherichia coli PPase (E-PPase). However, the pH optimum of MgPPi hydrolysis by V-PPase was shifted to more alkaline pH due to higher values of the pK a of ionizable groups for both the free enzyme and the enzyme-substrate complex. The stability of a hexameric form of V-PPase has been studied as a function of pH. The corresponding pK a of a group that controls the stability of the hexamer at pH below 6 (pK a = 4.4) was significantly lower than in the other hexameric PPases. The crystal structure reported here is analyzed and compared with the structure of E-PPase. The location of amino acid residues that differ in V-PPase and E-PPase is discussed. Since V-PPase has been found to retain its hydrolytic activity in high ionic strength media, the observed structural and kinetic features are analyzed in view of the possible osmoadaptation of this protein.
Keywords: pyrophosphatase; Vibrio cholerae ; osmoadaptation; X-ray analysis; crystal structure

Cloning and characterization of NAD-dependent protein deacetylase (Rv1151c) from Mycobacterium tuberculosis by Jing Gu; Jiao-Yu Deng; Ru Li; Hongping Wei; Zhiping Zhang; Yafeng Zhou; Ying Zhang; Xian-En Zhang (743-748).
Sir2 family proteins are highly conserved and catalyze a well-characterized NAD-dependent protein deacetylation reaction that regulates multiple cellular processes including aging, gene silencing, cellular differentiation, and metabolic pathways. Little is known about Sir2 family proteins in bacteria. The Sir2 homolog Rv1151c of Mycobacterium tuberculosis was cloned and over-expressed in Escherichia coli, and the protein then purified by Ni2+-affinity chromatography to homogeneity. The purified recombinant protein showed a typical NAD-dependent protein deacetylase activity that could be inhibited by nicotinamide and other known Sir2 inhibitors. The optimal temperature and pH for activity of Rv1151c are 25°C and pH 9 ± 1, respectively. Rv1151c is capable of deacetylating the acetyl-CoA synthetase from M. tuberculosis. However, unlike Sir2 family proteins identified from other bacteria, Rv1151c shows a substrate-independent NAD glycohydrolase activity in accordance with its auto-ADP ribosylation activity.
Keywords: ADP-ribosyltransferase; deacetylase; Mycobacterium tuberculosis ; NAD glycohydrolase; Sir2

Telomerase from yeast Saccharomyces cerevisiae is active in vitro as a monomer by D. M. Shcherbakova; K. A. Sokolov; M. I. Zvereva; O. A. Dontsova (749-755).
A system for isolation of yeast telomerase via RNA affinity tag in TLC1 RNA was developed. Streptavidin aptamer was inserted at two different positions in TLC1 RNA. Telomerase with TLC1 RNA with one of these inserts is functional in vivo and can be isolated by affinity chromatography in vitro. A telomerase preparation isolated using this technique from a strain producing two distinguishable TLC1 RNA molecules (with and without aptameric insertion) resulted in isolation of active telomerase containing only TLC1 RNA with the aptamer. Our results indicate that yeast telomerase is active in vitro as a monomer.
Keywords: yeast telomerase; RNA aptamer; telomerase dimerization

Lipid-protein nanodiscs: Possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides by Z. O. Shenkarev; E. N. Lyukmanova; O. I. Solozhenkin; I. E. Gagnidze; O. V. Nekrasova; V. V. Chupin; A. A. Tagaev; Z. A. Yakimenko; T. V. Ovchinnikova; M. P. Kirpichnikov; A. S. Arseniev (756-765).
High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). 15N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and 31P-NMR spectroscopy. The 2D 1H-15N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased 1HN line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. 15N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (∼40 nsec at 45°C) is indicative of additional peptide motions within the Aam-I/LPN complex.
Keywords: nanodisc; apolipoprotein; high-density lipoprotein particle; membrane protein; membrane-active peptide; model membranes; NMR spectroscopy

Competitive and complimentary relationships of various peroxidase substrates were studied to elucidate the enzymatic mechanisms underlying production of reactive oxygen species in plant cell apoplast. Dianisidine peroxidase released from wheat seedling roots was inhibited by ferulate and coniferol, while ferulic and coniferyl peroxidases were activated by o-dianisidine. Both ferulate and coniferol, when added together with hydrogen peroxide, stimulated superoxide production by extracellular peroxidase. We suggest that substrate-substrate activation of extracellular peroxidases is important for stress-induced oxidative burst in plant cells.
Keywords: wheat root; superoxide; peroxidase; phenol; extracellular solution

The effect of pyrophosphate (PPi) on labeled nucleotide incorporation into noncatalytic sites of chloroplast ATP synthase was studied. In illuminated thylakoid membranes, PPi competed with nucleotides for binding to noncatalytic sites. In the dark, PPi was capable of tight binding to noncatalytic sites previously vacated by endogenous nucleotides, thereby preventing their subsequent interaction with ADP and ATP. The effect of PPi on ATP hydrolysis kinetics was also elucidated. In the dark at micromolar ATP concentrations, PPi inhibited ATPase activity of ATP synthase. Addition of PPi to the reaction mixture at the step of preliminary illumination inhibited high initial activity of the enzyme, but stimulated its activity during prolonged incubation. These results indicate that the stimulating effect of PPi light preincubation with thylakoid membranes on ATPase activity is caused by its binding to ATP synthase noncatalytic sites. The inhibition of ATP synthase results from competition between PPi and ATP for binding to catalytic sites.
Keywords: ATP synthase; CF0CF1 ; coupling factor CF1 ; noncatalytic sites; chloroplasts

In the present study, two molecular forms of goat lung cystatin (GLC), I and II, were purified to homogeneity by a two-step procedure including ammonium sulfate precipitation (40–60%) and ion exchange chromatography. The inhibitor forms migrated as single bands under native and SDS-PAGE with and without reducing agent giving molecular mass of 66.4 and 76.4 kDa, respectively. GLC-I possesses 0.07% and GLC-II 2.3% carbohydrate content and no -SH groups. GLC-I showed greater affinity for papain than for ficin and bromelain. Immunological studies showed that the inhibitor was pure and there was cross reactivity between anti-GLC-I serum and goat brain cystatin. Both inhibitor forms were stable in the pH range of 3–10 and up to 75°C. GLC-I was found to possess 49% α-helical structure by CD spectroscopy. The inhibitor-papain complexes showed conformational changes as invoked by UV and fluorescence spectroscopic studies.
Keywords: goat lung cystatin; ion-exchange chromatography; kinetics; circular dichroism; ELISA; fluorescence spectroscopy

Cooperative binding of substrates to transketolase from Saccharomyces cerevisiae by I. A. Sevostyanova; V. A. Selivanov; V. A. Yurshev; O. N. Solovjeva; S. V. Zabrodskaya; G. A. Kochetov (789-792).
Catalytic activity of two active sites of transketolase and their affinity towards the substrates (xylulose-5-phosphate and ribose-5-phosphate) has been studied in the presence of Ca2+ and Mg2+. In the presence of Ca2+, the active sites exhibit negative cooperativity in binding both xylulose-5-phosphate (donor substrate) and ribose-5-phosphate (acceptor substrate) and positive cooperativity in the catalytic transformation of the substrates. In the presence of Mg2+, nonequivalence of the active sites is not observed.
Keywords: transketolase; nonequivalence of active sites; bivalent cations; xylulose-5-phosphate; ribose-5-phosphate

Features of structural organization and expression regulation of malate dehydrogenase isoforms from Rhodobacter sphaeroides strain 2R by A. T. Eprintsev; M. A. Klimova; K. D. Shikhalieva; D. N. Fedorin; M. T. Dzhaber; E. I. Kompantseva (793-799).
Two isoforms of malate dehydrogenase (MDH), dimeric and tetrameric, have been found in the purple non-sulfur bacterium Rhodobacter sphaeroides strain 2R, devoid of the glyoxylate shunt, which assimilate acetate via the citramalate cycle. Inhibitory analysis showed that the 74-kDa protein is involved in tricarboxylic acid cycle, while the 148-kDa MDH takes part in the citramalate pathway. A single gene encoding synthesis of the isologous subunits of the MDH isoforms was found during molecular-biological investigations. The appearance in the studied bacterium of the tetrameric MDH isoform during growth in the presence of acetate is probably due to the increased level of mdh gene expression, revealed by the real-time PCR, the product of which in cooperation with the citramalate cycle enzymes plays an important role in acetate assimilation.
Keywords: malate dehydrogenase; purification; isoforms; expression regulation

Purification and characterization of pectin lyase secreted by Penicillium citrinum by S. Yadav; P. K. Yadav; D. Yadav; K. D. S. Yadav (800-806).
The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K m and k cat values were found to be 1 mg/ml and 76 sec−1, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.
Keywords: pectin lyase; retting; solid state fermentation; Penicillium citrinum ; Cannabis sativa ; Crotalaria juncea ; Linum usitatissimum

The peripheral light-harvesting complex II (LHII) is an important component of the photosynthetic apparatus of Rhodobacter sphaeroides. In this study, genetic, biochemical, and spectroscopic approaches were applied to investigate the spectral properties and functions of LHII in which two amino acid residues Phe32 and Leu42 in the transmembrane helix domain of pucB-encoded β-apoprotein were replaced by Leu and Pro. The mutated LHII complex showed blue shift of absorbance peaks in the near infrared region at ∼801–845 nm in R. sphaeroides. It should be noted that the B800 peak was much lower than that of the native LHII, and transfer energy was efficient from the B800 to the B850 pigments in the LHII complex. The results suggest that the mutated pucB could be expressed in R. sphaeroides, and the functional LHII was assembled into the membrane of R. sphaeroides notwithstanding with the different spectral properties. These mutated residues were indeed critical for the modulation of characteristics and function of LHII complex.
Keywords: photosynthetic bacteria; light-harvesting complex II; Rhodobacter sphaeroides ; mutagenesis; expression analysis

Study of the inhibitory effect of fatty acids on the interaction between DNA and polymerase β by Jun Yang; Jing Yang; Zheng-Qin Yin; Jing Xu; Ning Hu; I. Svir; Min Wang; Yuan-Yi Li; Lei Zhan; Song Wu; Xiao-Lin Zheng (813-818).
The binding of human DNA polymerase β (pol β) to DNA template-primer duplex and single-stranded DNA in the absence or presence of pol β inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic acid, were used as potent pol β inhibitors. In the interaction between pol β and DNA, pol β could bind to ssDNA in a single binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol β inhibitors prevented the binding of pol β to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol β to the template-primer duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant inhibitory effect of nervonic acid on the pol β-duplex interaction was due to both a 2-fold decrease in the association rate and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate was observed, and the decrease in binding affinity of pol β to DNA was mainly due to 7-fold increase in the dissociation rate.
Keywords: DNA polymerase β; surface plasmon resonance; fatty acid; inhibition; SPR biosensor