The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
133 巻, 3 号
選択された号の論文の18件中1~18を表示しています
  • Kunio Kikuchi, Takuya Umehara, Kotaro Fukuda, Joonsung Hwang, Atsushi ...
    2003 年 133 巻 3 号 p. 263-270
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    The internal ribosome entry site (IRES) is important for translation of hepatitis C virus (HCV) mRNA and has a unique RNA structure containing conserved domains I to IV. To investigate the function of domain II, we selected RNA aptamers that bind to domain II of HCV IRES by applying a simple and convenient selection method using a hybridized tag for fixing domain II RNA on magnetic beads instead of synthesizing long RNA. In addition, we employed surface plasmon resonance (SPR) technology to measure the binding affinity of each generation and to obtain detailed kinetic constants. The selected aptamers have a consensus sequence, 5'-UAUGGCU-3', which is complementary to the apical loop of domain II. The loop-loop interaction between the consensus sequence and domain II was confirmed by mutagenesis and nuclease mapping analyses. Binding affinities were dependent on the local structure containing the conserved sequence. The aptamers could inhibit IRES-dependent translation.
  • Tetsuya Yoshida, Yuji Tsuruta, Makoto Iwasaki, Shoji Yamane, Takahiro ...
    2003 年 133 巻 3 号 p. 271-277
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    SRCL/CL-P1 was recently identified as a scavenger receptor with a C-type lectin domain, which was expressed in vascular endothelial cells and could bind to Grampositive and Gram-negative bacteria, yeast and oxidized LDL. We found that SRCL was expressed in some but not all nurse-like cells examined. Furthermore, to characterize the C-type lectin domain of SRCL, the secreted form of the C-type lectin domain (LEC-AP) of SRCL, which was fused to the signal sequence of IgG and alkaline phosphatase, was expressed in 293/EBNA-1 cells and the culture medium was used for the in vitro binding assay. LEC-AP specifically bound to GalNAc-conjugated gel in a Ca2+- dependent manner, and this binding was inhibited by free GalNAc, L-, D-fucose, D-galactose, lactose, and especially T antigen and Tn antigen. Furthermore, we examined whether or not SRcL could take up saccharide-conjugated particles. 293/EBNA-1 cells stably expressing SRcL were found to take up GalNAc but not mannose-conjugated particles on confocal microscopy. The binding of GalNAc-conjugated particles to these cells was quantitatively measured by comparing the x-means of individual cell populations. An approximately 2.1-fold increase in immunofluorescence intensity was observed for the SRCL transfectants compared to control vector transfectants. Our results provide a basis for understanding the scavenger function of SRCL as to carbohydrate-containing ligands.
  • Takashi Obama, Yukie Kan, Hiroh Ikezawa, Masayoshi Imagawa, Kikuo Tsuk ...
    2003 年 133 巻 3 号 p. 279-286
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    Bacillus cereus sphingomyelinase (SMase) is an extracellular hemolysin classified into a group of Mg2+-dependent neutral SMases (nSMase). Sequence compartison of bacterial and eukaryotic Mg2+-dependent nSMases has shown that several amino acid residues, including Glu-53 of B. cereus SMase, are conserved, suggesting a catalytic mechanism common to these enzymes. Mutational analysis has revealar that hemolytic and SM-hydrolyzing activities are abolished by E53A and E53Q mutations. Only the E53D mutant enzyme partially retains these activities, however, a significant devrease in the apparent kcat/Km for SM hydrolysis is observed by this mutation. Mg2+activates the wild-type enzyme in a two-step manner, i.e., at least two binding sites for Mg2+, high-and low-affinity, are present on the enzyme. The binding affinity of essential Mg2+ for the high-affinity site is decreased by the mutation. In addition, the binding affinities of Mn2+ and Co2+ (substitutes for Mg2+) are also decreased. On the contrary, the inhibitory effects of Ca2+, Cu2+, and Zn2+ on SM-hydrolyzing activity are not influenced by the mutation. The results indicate that Glu-53 of B. cereus SMase acts as a ligand for Mg2+ and is involved in the high-affinity Mg2+-binding site, which is independent of the binding site for inhibitory metals.
  • Fathy M. El-Fasakhany, Keiko Ichihara-Tanaka, Kenji Uchimura, Takashi ...
    2003 年 133 巻 3 号 p. 287-293
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the C-6 position of non-reducing GlcNAc. Human GlcNAc6ST-1 was expressed as a fusion protein with protein A in an insect cell line (Tn 5 cells) using the baculovirus system. The recombinant enzyme was purified to homogeneity by IgG Sepharose column chromatography. The substrate specificity and the kinetic properties of the enzyme were similar to those of the enzyme expressed in the mammalian system. The purified recombinant enzyme was used to synthesize 6-sulfo GlcNAcβ1-3Galβ1-4Glc, which was identified by time of flight mass spectrometry. This sulfated trisaccharide served as a better substrate for microsomal galactosyltransferase from the mouse colon compared to 6-sulfo GlcNAc. The purified recombinant enzyme was also used to sulfate oligosaccharide chains on fibrinogen after enzymatic desialylation and degalactosylation to expose nonreducing GlcNAc residues. It is known that desialylation greatly increases the rate of clotting of fibrinogen after the addition of thrombin. Subsequent sulfation of desialylated and degalactosylated fibrinogen slightly decreased the rate of clotting. The recombinant GlcNAc6ST-1 is a useful reagent for 6-sulfate exposed GlcNAc residues both in oligosaccharides and in glycoproteins.
  • Hideaki Nanamiya, Emiko Shiomi, Mitsuo Ogura, Teruo Tanaka, Kei Asai, ...
    2003 年 133 巻 3 号 p. 295-302
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    ComK protein of Bacillus subtilis positively regulates the transcription of several late competence genes as well as comK itself. We constructed a clpX disrupted mutant of B. subtilis and studied its effect on the regulation of ComK activation. When Pspac, which controls the comK gene in a multicopy plasmid, was induced by the addition of IPTG, comK transcripts were detected in both the clpX mutant and the wild type. However, the ComK protein could not be detected in the clpX disrupted mutant. To obtain further information, we constructed several comK-lacZ translational fusions covering different lengths of the comK gene, whose transcription is controlled by an IPTG inducible Pspac promoter. We found that both the expression of comK-lacZ directed β-galactosidase and the accumulation of ComK-LacZ fused protein, derived from the fusion containing the entire comK open reading frame, were extremely reduced in the clpX mutant compared with the wild type, while the accumulation of comK-lacZ transcripts in the clpX mutant after the addition of IPTG was about half that in the clpX+background. On the other hand, transcription, translation and activity of comK-lacZ were detected in both the clpX mutant and the wild type when the comK-lacZ fusion lacking the 3' region of the comK gene was induced. These results indicate that ClpX plays an important role in the regulation of ComK at the post-transcriptional level.
  • Yuji Satoh, Tetsuji Nakagawachi, Hisaya Nakadate, Yasuhiko Kaneko, Zen ...
    2003 年 133 巻 3 号 p. 303-308
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    WT1 at 11p13 is a tumor suppressor gene, an aberration of which causes Wilms' tumor (WT). Since WT1 expression is reduced in a certain poroportion of WTs and its mutation is found only in 10-20% of WTs, we examined WT1 gene silencing due to epigenetic alteration in a total of 22 WTs. WT1 expression was significantly reduced in half of WTs without any mutation in the WT1 gene itself, suggesting that the reduction of expression was possibly epigenetic. We found promoter hypermethylation in one WT with loss of heterozygosity (LOH) and showed that promoter methylation reduced reporter gene activity by a reporter assay. These data suggested that methylation was an epigenetic mechanism leading to WT1 silencing and that the expression-reduced allele by hypermethylation combined with LOH was consistent with the revised twohit model. In addition, as the β-catenin mutation is frequently associated with the WT1 mutation, the association of WT1 silencing with the β-catenin mutation was also investigated. β-catenin mutated in only one WT without WT1 silencing, suggesting that the β-catenin mutation was not associated with the reduction of WT1 expression.
  • Miroslawa Z. Barciszewska, Eliza Wyszko, Rolf Bald, Volker A. Erdmann, ...
    2003 年 133 巻 3 号 p. 309-315
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    This paper reports that the D-loop sequence of cellular mammalian ribosomal 5S RNAs is a natural leadzyme that specifically binds and cleaves in trans other RNA molecules in the presence of lead. The D-loops of these 5S rRNAs are similar in sequence to the active site of the leadzyme derived from tRNAPhe, which cleaves a single bond in cis. We have devised a 12 nt model substrate based on the leadzyme sequence cleaved in trans by a 12 nt RNA molecule containing of the D-loop sequence. The model reaction occurs only at the appropriate concentration of lead and enzyme/ substrate stoichiometry. The native 5S rRNA carries the same cleavage activity, although with different optimal lead concentration and stoichiometry. On the other hand, the isolated D-loop does not serve as a substrate when incubated with an RNA molecule with the potential to base pair with it and form the same internal loop (the bubble) present in the leadzyme-substrate complex. We show that the leadzyme cuts C-G, but not G-G or U-G linkages. The 5S rRNA leadzyme appears to have the shortest asymmetric pentanucleotide purine-rich loop flanked by two short double stranded RNAs. The leadzyme activity of native 5S rRNA may be an important aspect of lead toxicity in living cells. Because the leadzyme motif has been found in natural RNA species, its activity can be expressed in vivo even at a very low lead concentrations, of lead leading to the inactivation of other cellular RNAs. This might be one of the ways in which lead poisoning manifests itself at the molecular level. Lead toxicity is based not only on its binding to calcium and zinc binding proteins (such as Zn-fingers) and random hydrolysis of nucleic acids, but also, and most importantly, on the induction of the hydrolytic properties of RNA (RNA catalysis).
  • Masayasu Takada, Yoshinori Nakagawa, Mikio Yamamoto
    2003 年 133 巻 3 号 p. 317-324
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    On screening for microorganisms in soil obtained in Japan that produce large amounts of γ-cyclodextrin (γ-CD), we identified a novel alkalophilic bacterium, Bacillus clarkii 7364. The cyclodextrin glucanotransferase (CGTase) secreted into the culture medium by this bacterium was purified by affinity chromatography on a γ-CD-immobilized column, followed by chromatography on a gel filtration column. The enzyme converted 13.7% of pre-gelatinized potato starch (10% w/w per reaction mixture) into CDs, and the majority (79%) of the product CDs was of the γ form. This property is quite unique among known CGTases and thus we named this enzyme γCGTase. The N-terminal and internal amino acid sequences of γ-CGTase were determined and used to design PCR primers for amplification of the nucleotide sequence that encodes the γ-CGTase gene. The entire gene sequence amplified by PCR was determined and then cloned into E. coli. The recombinant enzyme synthesized by E. coli retained biochemical properties quite similar to those of the original one. Comparison of the deduced amino acid sequence of γ-CGTase with those of other known CGTases that have different product specificities revealed the importance of subsites -3 and -7 for the preferential γ-cyclization activity.
  • Kei Suga, Tetsuo Yamamori, Kimio Akagawa
    2003 年 133 巻 3 号 p. 325-334
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    HPC-1/syntaxin 1A is a member of the syntaxin family, and functions at the plasma membrane during membrane fusion as the target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (t-SNARE). We identified the membrane-anchor-ing region of HPC-1/syntaxin lA, and examined its role in anchoring of a protein to the plasma membrane. A series of mutants was created from a cysteine-less mutant of HPC-l/syntaxin 1A by substitution of each residue at the C-terminus with cysteine. The accessibility of the thiol-groups in each mutant was analyzed in vivo. The cysteine (C145) within the N-terminal cytosolic segment was labeled, but not that at C271 or C272, or any of those introduced at the C-terminus. The addition of additional residues to the C-terminal tail of HPC-1/syntaxin lA allowed labeling by thiol-specific reagents. A monoclonal antibody directed against the C-terminal tail peptide did not react with the protein located at the plasma membrane. In addition, subcellular fractionation and immunocytochemical analyses with various transmembrane mutants showed that the C-terminal tail comprising eight amino acids is essential for anchoring of HPC-l/syntaxin 1A to the plasma membrane. These results indicate that the Cterminal membrane-anchoring region, which comprises 23 amino acids, does not traverse the lipid-bilayer and that the C-terminal tail is essential for anchoring of HPC-1/syntaxin lA to the plasma membrane.
  • Kei Odai, Tohru Sugimoto, Minoru Kubo, Etsuro Ito
    2003 年 133 巻 3 号 p. 335-342
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    Even though glutamic acid contains only one more carboxyl group than γ-aminobu-tyric acid (GABA), these neurotransmitters are recognized by their own specific receptors. To understand the ligand-recognition mechanism of the receptors, we must determine the geometric and electronic structures of GABA and glutamic acid in aqueous conditions using the ab initio calculation. The results of the present study showed that the stable structure of GABA was the extended form, and it attracted both cations and anions. Glutamic acid only attracted cations and was stabilized in four forms in aqueous conditions: Type 1 (an extended form), Type 2 (a rounded form), and Types 3 and 4 (twisted forms of Type 1). The former two types had low energy and the energy barrier between them was estimated to be small. These results showed that most free glutamic acid is present as Type 1, Type 2, and transient forms. The present results therefore suggest that the flexibility of the geometric structures of ligands should be taken into account when we attempt to elucidate the mechanism of recognition between ligands and receptors, in addition to the physicochemical characteristics of liErands and receptors.
  • Young-Ae Lee, Soomin Lee, Hyun Mee Lee, Chong-Soon Lee, Seog K. Kim
    2003 年 133 巻 3 号 p. 343-349
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    When meso-tetrakis(3-N-methylpyridiniumyl) porphyrin (m-TMPyP) formed a complex with poly[d(A-T)2], an intense bisignate excitonic CD in the Soret absorption region was observed. The excitonic CD of the m-TMPyP-poly[d(A-T)2] complex is unique in that no other combination of the related porphyrin, namely, meso-tet-rakis(n-N-methylpyridiniumyl)porphyrin (where n=2, 4), and polynucleotide including calf thymus DNA, poly[d(G-C)2], poly[d(I-C)2], and poly(dA)⋅poly(dT), exhibits a comparable CD spectrum. From the [drug]/[DNA] ratio-dependence of the intensity and the shape of the CD spectrum, this porphyrin species is assigned to an exten-sively aggregated form. The extensively aggregated porphyrin disperses in 1 h after mixing to form moderately stacked porphyrin at a low mixing ratio. The magnitude of linear dichroism of the extensively aggregated porphyrin was small and the sign was negative in the Soret band, which indicated that the molecular plane of porphyrin in the complex is strongly tilted. On the other hand, the molecular plane of porphyrin is almost parallel to the DNA base plane (perpendicular to the DNA helix axis) in the moderately stacked form.
  • Satoru Sekiya, Fumiko Nishikawa, Kotaro Fukuda, Satoshi Nishikawa
    2003 年 133 巻 3 号 p. 351-359
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    RNA aptamers that bind specifically to hepatitis C virus (HCV) NS3 protease domain (ΔNS3) were identified in previous studies. These aptamers, G9-I, -II, and -III, were isolated using an in vitro selection method and they share a common loop with the sequence 5'-GA(A/U)UGGGAC-3'. The aptamers are potent inhibitors of the NS3 protease in vitro and may have potential as anti-HCV compounds. G9-I has a 3-way stemloop structure and was selected for further characterization using site-directed mutagenesis. Mutations or deletions in stem-loop II do not interfere with binding or inhibition of ΔNS3, but mutations or deletions in stem I and stem-loop III destroy the G9-I active conformation and interfere with inhibition of NS3 protease. A 51 nt frag-ment of 74 nt G9-I was identified (ΔNEO-III) as is the minimal fragment of G9-I that is an effective inhibitor of the NS3 protease. Tertiary interactions involving functionally important nucleotides were identified in the active structure of G9-I using nucleotide analog interference mapping (NAIM). Strong interferences were focused in the conserved loop involving stem-loop III and stem I. For example, analog-interference caused at A(+8) and C(+24)-G(-36) base pair implied an A-minor motif involving the intramolecular base triple A(+8)•C(+24)-G(-36), which is further supported by muta-genesis. These results suggested the interaction of stem I and stem-loop III is essential for the function of G9-I aptamer.
  • Masakatsu Fujinoki, Takeshi Kawamura, Toshifusa Toda, Hideki Ohtake, T ...
    2003 年 133 巻 3 号 p. 361-369
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    In our previous paper [M. Fujinoki et at. (2001) Biomed. Res. 22, 45-58], we reported that two types of 36-kDa protein, which were designated as 36K-A protein and 36K-B protein, obtained from hamster sperm flagella were phosphorylated at serine residues associated with the regulation of motility activation. In the present experiments, it was suggested that these two types of 36-kDa protein were phosphorylated in a cAMP-dependent manner associated with motility activation of hamster spermatozoa. Because the 36K-B protein was the most intensely phosphorylated in a cAMP-dependent manner, attempts were made to further characterize it. The 36K-B protein was assumed to be localized in the middle piece. The localization of the 36K-B protein was the same as that of the 36-kDa protein reported in our previous paper [Y. Si et al. (1999) Mol. Reprod. Dev. 52, 328-334]. In order to identify the 36K-B protein, it was analyzed by peptide mass finger printing and amino acid sequencing. The results suggested that the 36K-B protein was a pyruvate dehydrogenase E1 component β subunit and a component of the mitochondrial sheath of the middle piece.
  • Kenji Kashiwagi, Kiyotaka Shiba, Kaoru Fukami-Kobayashi, Tetsuo Noda, ...
    2003 年 133 巻 3 号 p. 371-376
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    The family of periplasmic binding proteins (PBPs) is believed to have arisen from a common ancestor and to have differentiated into two types. At first approximation, both types of PBPs have the same fold pattern, reflecting their common origin. How-ever, the connection between the main chains of a type 2 PBP is more complicated than a type 1 PBP's. We have been interested in the possibility that such structural changes affect the folding of PBPs. In this study, we have characterized the folding pathways of MglB (a type 1 PBP) and ArgT (a type 2 PBP) by using urea gradient gel electrophoresis, fast protein size-exclusion liquid chromatography and hydrophobic dye ANS binding assay. We found a distinct difference in folding between these two proteins. The folding of Mg1B followed a simple two-state transition model, whereas the folding of ArgT was more complicated.
  • Kouichi Mogi, Haruo Takeshita, Toshihiro Yasuda, Tamiko Nakajima, Emik ...
    2003 年 133 巻 3 号 p. 377-386
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17, 000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Va167, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.
  • Takeshi Nakanishi, Hideo Inoue, Masaya Kitamura
    2003 年 133 巻 3 号 p. 387-393
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
    We identified the SOD gene in the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F) and constructed a high-level expression system in Escherichia coli. A 2.6-kbp DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with EcoRI and SmaI contained the SOD gene and part of another open reading frame. The amino acid sequence deduced from the SOD gene, which was composed of 238 amino acid residues, showed high homogeneity with iron-containing SOD (Fe-SOD) and predicted that the amino terminus of this protein would carry an export signal peptide. We produced the precursor form of SOD (PSOD) and the mature form of SOD (MSOD), which lacked the putative signal peptide. In E. coli, PSOD was present in insoluble inclusion bodies, and its putative signal peptide was not cleaved. In contrast, MSOD contained one iron per mononer and formed a dimer, which exhibited an SOD activity of 850U/mg. Furthermore, D. vulgaris soluble extract showed a band of SOD activity on native polyacrylamide gel that migrated to the same point as MSOD. The intracellular localization of SOD and its role in D. vulgaris are also discussed.
  • 2003 年 133 巻 3 号 p. 395a
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
  • 2003 年 133 巻 3 号 p. 395b
    発行日: 2003年
    公開日: 2008/06/30
    ジャーナル フリー
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