巻号一覧
129 巻, 3 号
選択された号の論文の20件中1～20を表示しています
- |<
- <
- 1
- >
- >|
-
Kazuya Taniguchi, Shunji Kaya, Kazuhiro Abe, Sven Mårdh2001 年 129 巻 3 号 p. 335-342
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーSince the discovery of Na/K-ATPase, evidence has accumulated to suggest that 1 mol of ATP hydrolysis occurs via the Na+-occluded ADP-sensitive phosphoenzyme, the K+-sensitive phosphoenzyme and the K+-occluded enzyme accompanying active transport of 3Na+ and 2K+ according the Post-Albers scheme. However, some controversial issues have arisen concerning whether the functional unit of the enzyme is an αβ-protomer or a much higher oligomer, which would be related to the mechanism of transport, either sequential or simultaneous. Detailed studies of oligomer interaction and the reactivity of the enzyme and a comparison of the extent of phosphorylation with ligand-binding capacities in the presence or absence of ATP hydrolysis and others strongly suggest that the functional unit of the enzyme in the membrane is a tetraprotomer, (αβ)4. They also suggest that each reaction intermediate of the Post-Albers scheme, respectively, reflects half of the site property of the intermediate and that another half binds ATP. These data may be useful not only to answer the long-standing question of whether the mechanism functions in the presence of both Na+ and K+ but also contribute to a better understanding of the mechanism of P-type pump ATPase in general.抄録全体を表示PDF形式でダウンロード (863K) -
Hiroshi Itou, Isao Tanaka2001 年 129 巻 3 号 p. 343-350
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThe Escher ichia coli DNA-binding protein OmpR is the best characterized of those regulator proteins making up “two-component system, ” the simplest known form of bacterial signal transduction systems. Previous inspections of the E. coil genome DNA sequences have revealed that there are 15 proteins whose amino acid sequences show extensive similarities to that of OmpR (the OmpR-family of proteins). The three-dimensional structures of several OmpR-family proteins have been determined. In this review, we investigated the structures and amino acid sequences of this family of proteins. The results reveal several notable conservative varieties in their tertiary structures and functions.抄録全体を表示PDF形式でダウンロード (3226K) -
Yoshio Kimura, Yuki Yamanishi, Yushi Tokumasu, Hideaki Terasaka, Junko ...2001 年 129 巻 3 号 p. 351-356
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThe mac-1 gene of Myxococcus xanthus TA, an antibiotic TA producer, encoded a protein with strong sequence similarity to the antibiotic ATP-binding cassette (ABC) transporter for macrolide antibiotics. The mac-1 gene encoding protein (Mac-1) had two ATP-binding domains containing Walker A and B motifs, and no hydrophobic transmembrane regions. Insertional inactivation of mac-1 caused enhanced sensitivity to oleandomycin, a macrolide antibiotic, while the mac-1 mutant showed normal export of antibiotic TA into the extracellular fluid. The mac-1 mutant could form mounds, but was unable to form fruiting bodies or sporulate under nutrient starvation. A primary role for Mac-1 in M. xanthus may be as a transporter which exports or imports a molecule required for the sporulation process.抄録全体を表示PDF形式でダウンロード (1233K) -
Masaya Kitamura, Takeshi Nakanishi, Shuichi Kojima, Izumi Kumagai, Hid ...2001 年 129 巻 3 号 p. 357-364
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーWe identified a gene encoding a catalase from the anaerobic bacteria Desulfovibrio vulgaris (Miyazaki F), and the expression of its gene in Escherichia coli. The 3.3-kbp DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with EcoRI and SaiI was found to produce a protein that binds protoheme IX as a prosthetic group in E. coli. This DNA fragment contained a putative open reading frame (Kat) and one part of another open reading frame (ORF-1). The amino acid sequence of the amino terminus of the protein purified from the transformed cells was consistent with that deduced from the nucleotide sequence of Kat in the cloned fragment of D. vulgaris (Miyazaki F) DNA, which may include promoter and regulatory sequences. The nucleotide sequence of Kat indicates that the protein is composed of 479 amino acids per monomer. The recombinant catalase was found to be active in the decomposition of hydrogen peroxide, as are other catalases from aerobic organisms, but its Km value was much greater. The hydrogen peroxide stress against D. vulgaris (Miyazaki F) induced the activity for the decomposition of hydrogen peroxide somewhat, so the catalase gene may not work effectively in vivo.抄録全体を表示PDF形式でダウンロード (1422K) -
Kaoru Konishi, Shin-ichiro Kojima, Tsuyoshi Katoh, Michio Yazawa, Kazu ...2001 年 129 巻 3 号 p. 365-372
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーPrevious studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of ΔMID lacked the motor domain, that of Δ(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCIconcentration became less than 0.1M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of ΔMD and Δ(MD+ELC), but that of singleheaded HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of ΔMD and Δ(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.抄録全体を表示PDF形式でダウンロード (2300K) -
Satoru Yamaguchi, Satoru Tuzi, Koka Yonebayashi, Akira Naito, Richard ...2001 年 129 巻 3 号 p. 373-382
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーWe have recorded 13C NMR spectra of [2-13C]-, [1-13C]-, [3-13C], - and [l, 2, 3-13C3] Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The 13C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-13C]-, [1-13C]Ala-, and [1-13C] G lylabeled bR, due to the presence of conformational fluctuation with correlation times of 10-4 s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or μs in the surface loops of membrane proteins. Instead, the three well-resolved 13C CP-MAS NMR signals of [2-13C] Ala-bR, at 50.38, 49.90, and 47.96ppm, were ascribed to the C-terminal α-helix previously proposed from the data for [3-13C] Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such 13C NMR signals were substantially broadened when 13C NMR spectra of fully labeled [1, 2, 3-13C] Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of 13C-13C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among 13C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal α-helix, loops, and transmembrane α-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.抄録全体を表示PDF形式でダウンロード (2103K) -
Laura Matkovic, Celso E. Gomez-Sanchez, Eduardo N. Cozza2001 年 129 巻 3 号 p. 383-390
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーA simple mathematical model for studying mechanism-based inhibitors (MBIs) is presented. The mathematical equations are deduced for an experimental protocol consist ing of a first incubation of the enzyme in the presence of MBI followed by a washing protocol to eliminate free MBI. Finally enzyme activity (initial velocity) is measured with specific substrate. The representation of the final equation obtained is a straight line, and the MBI-specific association constant of velocity (k) can be calculated from its slope. The mathematical model was then challenged with the effect of 18-ethynyl-11-deoxycorticosterone (18-EtDOC) as an MBI on aldosterone biosynthesis from 11-deoxycorticosterone (DOC) in rat adrenal mitochondria. The last step of the mitochondrial biosynthesis of aldosterone consists of the conversion of DOC into corticosterone (B) or 18-hydroxy-11-deoxycorticosterone (18-OHDOC), and both steroids can then be transformed into aldosterone. The k (mM-1 min-1) values obtained for 18-EtDOC were: 451±36 for DOC to aldosterone; 177±16 for B to aldosterone; 175±15 for 18-OHDOC to aldosterone; and 2.7±0.2 for DOC to B. These results show that this MBI practically does not affect the metabolism of DOC to B in our enzyme preparation and that conversions of B and 18-OHDOC into aldosterone are catalyzed by the same enzyme.抄録全体を表示PDF形式でダウンロード (659K) -
Masao Teramoto, Kazuko Nakamasu, Mitsuhide Noshiro, Yoichi Matsuda, Os ...2001 年 129 巻 3 号 p. 391-396
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーDEC1/BHLHB2 is a novel cAMP-inducible basic helix-loop-helix (bHLH) transcriptional factor isolated from human chondrocyte cultures by the subtraction method [Shen et al. (1997) Biochem. Biophys. Res. Commun. 236, 294-298]. DEC1 seems to be involved in controlling the proliferation/differentiation of some cell lineages. We determined the structure of the human DEC1 gene and its chromosomal locus. Phylogenetic analysis and comparison of the gene structure showed that the DEC1 protein is a member of a new subgroup of the proline bHLH protein family that diverged earlier than other proline bHLH proteins including HES, hairy and E (spl). The human DEC1 gene spans approximately 5.7kb and contains 5 exons. The putative promoter region contains multiple GC boxes but no TATA box. A primer extension study showed multiple transcriptional initiation sites. In the 5'-flanking region of the DEC1 gene, several transcriptional factor binding sites, including a CAMP-responsive element (CRE), were found using the transcription factor database. The DEC1 gene locates at Chromosome 3p25.3-26 by the FISH method. This is the first study to determine the genomic structure of the DEC1 gene subgroup.抄録全体を表示PDF形式でダウンロード (1672K) -
Shiro Kitaura, Koji Suzuki, Shigeyuki Imamura2001 年 129 巻 3 号 p. 397-402
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーMonoacylglycerol lipase [MGLP, EC 3.1.1.23] is produced intracellularly by the moderately thermophilic Bacillus sp. strain H-257. The gene encoding MGLP was cloned, sequenced, and expressed in Escherichia coli. A genomic library of Bacillus sp. strain H-257, prepared in the plasmid vector pACYC184, was screened with a 0.2-kbp DNA fragment amplified by the polymerase chain reaction (PCR) with oligonucleotide primers designed based on the amino acid sequence of a purified MGLP. The plasmid pMGLP31, identified by hybridization with the amplified DNA fragment, contained a 5.3-kbp insert from Bacillus sp. strain H-257 DNA. Sequence analysis of the MGLP gene revealed an open reading frame encoding MGLP consisting of 250 amino acids, with a calculated molecular mass of 27.4 kDa. The deduced amino acid sequence of MGLP contained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conserved among lipases, esterases, and serine proteases. The MGLP is homologous to a putative esterase/lipase from Streptomyces coelicolor (41.8% homology). When pMGLP31 was introduced into E. coli DH1, the transformants produced MGLP intracellularly as an active form to an approximately 13.8-fold greater extent than Bacillus sp. strain H-257. The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic behavior, isoelectric point, and physicochemical and catalytic properties.抄録全体を表示PDF形式でダウンロード (1182K) -
Seiji Kosuge, Toshiro Maekawa, Chisato Saito, Takashi Tanaka, Isao Kou ...2001 年 129 巻 3 号 p. 403-409
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThe inhibitory effects of galloyl pedunculagin (GP) and eugeniin on the phosphorylation of histone H2B by cAMP-dependent protein kinase (A-kinase) and autophosphorylation of its β-regulatory subunit (A-kinase β) were examined in vitro. It was found that (i) GP (ID50=approx. 50nM) effectively inhibits the activity of A-kinase (heterodimer), but high doses are required to inhibit the activities of the α-catalytic subunit (ID50=approx. 0.25μM) and casein kinase II (CK-II, ID50=approx. 0.6μM); (ii) GP inhibits the autophosphorylation of A-kinase β in a dose-dependent manner with an ID50 of approx. 6.6nM, which is about 30-fold lower than that observed with CK-IIβ; and (iii) GP reduces the suppressive effect of the β-subunit on the activity of the α-subunit. In addition, purified bovine heart A-kinase precipitates when incubated with excess GP at pH 5.0. A similar precipitation of A-kinase was observed with eugeniin. These results show that the direct binding of GP to the β-subunit prevents the physiological interaction between the β-and α-subunits of A-kinase in vitro. This conclusion is presumably consistent with the binding affinity of proline-rich proteins with tannins, since A-kinase β contains a proline-rich domain that interacts with GP or eugeniin. Therefore, GP will serve as a powerful inhibitor for in vitro and in vivo cellular studies of A-kinase β-mediated signal transduction.抄録全体を表示PDF形式でダウンロード (3010K) -
Ling Yu, Tetsuo Ishida, Kiyoshi Ozawa, Hideo Akutsu, Kihachiro Horiike2001 年 129 巻 3 号 p. 411-421
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーTwo distinct forms of acetate kinase were purified to homogeneity from a sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F. The enzymes were separated from the soluble fraction of the cells on anion exchange columns. One acetate kinase (AK-I) was a homodimer (αs2) and the other (AK-II) was a heterodimer (αsαL). On SDS-PAGE, αL and αS subunits migrated as bands of 49.3 and 47.8 kDa, respectively, but they had an identical N-terminal amino acid sequence. A rapid HPLC method was developed to directly measure ADP and ATP in assay mixtures. Initial velocity data for AK-I and AK-II were collected by this method and analyzed based on a random sequential mechanism, assuming rapid equilibrium for the substrate binding steps. All kinetic parameters for both the forward acetyl phosphate formation and the reverse ATP formation catalyzed by AK-I and AK-II were successfully determined. The two enzymes showed similar kinetic properties in Mg2+ requirement, pH-dependence and magnitude of kinetic parameters. These results suggest that two forms of acetate kinase are produced to finely regulate the enzyme function by post-translational modifications of a primary gene product in Desulfovibrio vulgaris.抄録全体を表示PDF形式でダウンロード (2932K) -
Shin Kondo, Akashi Ohtaki, Takashi Tonozuka, Yoshiyuki Sakano, Shigehi ...2001 年 129 巻 3 号 p. 423-428
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーCrystals of the mutant E354A of Thermoactinomyces vulgaris R-47 α-amylase 2 (TVAII) complexed with β-cyclodextrin were prepared by a soaking method, and the diffraction data were collected at 100 K, using Synchrotron radiation (SPring-8). The crystals belong to an orthorhombic system with the space group P212121 and cell dimensions a=111.1 Å, b=117.7 Å, c=113.3 Å, which is almost isomorphous with crystals of the wildtype TVAII, and the structure was refined to an R-factor=0.208 (Rfree=0.252) using 3.0 Å resolution data. The refined structure shows that the interactions between Phe286 and two C6 atoms of β-cyclodextrin at the hydrolyzing site are important for TVAII to recognize cyclodextrins as substrates. This observation from the X-ray structure was supported by kinetic analyses of cyclodextrins using the wild-type TVAII, the mutant F286A and F286L. These studies also suggested that the TVAII-hydrolyzing mechanism for cyclodextrins is slightly different from that for starch.抄録全体を表示PDF形式でダウンロード (1560K) -
Hiroshi Oneda, Kuniyo Inouye2001 年 129 巻 3 号 p. 429-435
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThe effects of the metalloproteinase inhibitors thiorphan and R-94138 on the matrilysincatalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2, 4-dinitrophenyl)-L-2, 3-diamino-propionyl]-L-Ala-L-Arg-NH2 [MOCAc-PLGL(Dpa)AR] were examined. The inhibitor constants (Ki) of thiorphan and R-94138 for matrilysin at pH 7.5, 25°C were determined to be 11.2 and 7.65μM, respectively. From the temperature dependence of the Ki values at pH 7.5, the standard enthalpy change (ΔH°) values for the binding of matrilysin with thiorphan and R-94138 were determined to be -(18.2±0.9) and (1.65±1.07) kJ•mol-1, respectively. The binding of matrilysin to thiorphan is exothermic and the free energy change in the complex formation depends mainly on the change in enthalpy, while the binding to R-94138 is endothermic and typically entropy-driven. Hydrophobic interactions are suggested to contribute significantly to the binding of matrilysin to R-94138 as well as to the substrate. The pH dependence of the Ki value suggests that at least two ionizing groups with pKa values of 4.5 and 9.1-9.3 are involved in the binding. The matrilysin activity is regulated by ionizing groups with pKa values of 4.3 and 9.6. Both inhibition and hydrolysis are suggested to be controlled by the same residues in matrilysin, most likely Glu 198 and Tyr 219, respectively.抄録全体を表示PDF形式でダウンロード (645K) -
Takuya Numata, Tsuyoshi Katoh, Michio Yazawa2001 年 129 巻 3 号 p. 437-444
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーSmooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by et-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased Km for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing Km but only slightly increased Km for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the absence of Ca2+/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca2+/CaM-dependent activation.抄録全体を表示PDF形式でダウンロード (2546K) -
Takumasa Nishimura, Kei Horino, Hiroshi Nishiura, Yoko Shibuya, Takehi ...2001 年 129 巻 3 号 p. 445-454
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーA pancreatic carcinoma cell line, AsPC-l, underwent apoptosis in vitro when heattreated for 60min at 43°C. Apoptotic AsPC-1 cells liberated a monocyte chemotactic factor into the culture supernatant 24 to 30h after the heat-treatment. This factor was immunologically identified as the cross-linked homodimer of S19 ribosomal protein (RP S19), since the majority of the chemotactic activity was absorbed by both anti-RP S19 rabbit antibodies and an anti-isopeptide bond monoclonal antibody immobilized on agarose beads. Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24h after the heat-treatment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The chemotactic activity acquirement as well as the transglutaminase activity were blocked by treatment of the extract with anti-type II transglutaminase rabbit antibodies. When the recombinant RP S19 was treated with an authentic type II transglutaminase, the dimerization of RP S19 concomitant with the generation of the monocyte chemotactic activity was observed. Peptide-map analyses involving amino acid sequencing demonstrated that the inter-molecular isopeptide bond was heterogenous: G1n12 or G1n137 and Lys29 or Lys122 were cross-linked. Site-directed mutagenic analysis indicated that the cross-linking of Gln137, but not other residues such as Gln12, Lys29, and Lys122, was essential for expression of the chemotactic activity.抄録全体を表示PDF形式でダウンロード (2080K) -
Koji Tomoo, Kazuhiro Satoh, Yuko Tsuda, Keiko Wanaka, Shosuke Okamoto, ...2001 年 129 巻 3 号 p. 455-460
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーPKSI-527 is a noncovalent-type low-molecular-weight inhibitor. The X-ray crystal structure of the trypsin-PKSI-527 complex revealed a binding mode (Form II) different from the previously reported one (Form I) [Nakamura, M. et al. (1995) Biochem. Biophys. Res. Commun. 213, 583-587]. In contrast to the previous case, the electron density of the inhibitor revealed the whole structure clearly. Each structural part of the inhibitor in Forms I and II was differently located at the active site, although the modes of binding of the terminal amino group to the Asp189 carboxyl group were similar. This binding diversity, which is a characteristic of the noncovalent-type low-molecular-weight inhibitor, provides a suitable example for estimating the possible mechanism toward the enzymatic inhibition, together with the structural basis necessary for a specific inhibitor. The mode of binding in Form II reflects the inhibitor-specific situation and is in contrast with the substrate-mimetic binding mode for Form I. Based on the generally accepted catalytic mechanism for serine protease, we propose that a water molecule located at the catalytic site plays an important role in blocking the catalytic function of the reactive Ser193 OH group.抄録全体を表示PDF形式でダウンロード (399K) -
Duvjir Suvd, Zui Fujimoto, Kenji Takase, Masatoshi Matsumura, Hiroshi ...2001 年 129 巻 3 号 p. 461-468
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーThe crystal structure of a thermostable e-amylase from Bacillus stearothermophilus (BSTA) has been determined at 2.0 Å resolution. The main-chain fold is almost identical to that of the known crystal structure of Bacillus licheniformis α-amylase (BLA). BLA is known to be more stable than BSTA. A structural comparison between the crystal structures of BSTA and BLA showed significant differences that may account for the difference in their thermostabilities, as follows. (i) The two-residue insertion in BSTA, Ile181-Gly182, pushes away the spatially contacting region including Asp207, which corresponds to Ca2+-coordinating Asp204 in BLA. As a result, Asp207 cannot coordinate the Ca2+, (ii) BSTA contains nine fewer hydrogen bonds than BLA, which costs about 12 kcal/mol. This tendency is prominent in the (β/α)8.-barrel, where 10 fewer hydrogen bonds were observed in BSTA. BLA forms a denser hydrogen bond network in the interhelical region, which may stabilize α-helices in the barrel. (iii) A few small voids observed in the a-helical region of the (β/α)8.-barrel in BSTA decrease inter-helical compactness and hydrophobic interactions. (iv) The solvent-accessible surface area of charged residues in BLA is about two times larger than that in BSTA.抄録全体を表示PDF形式でダウンロード (2982K) -
Jong-Moon Kim, Fabrice Maraboeuf, Seog K. Kim, Akira Shinohara, Masayu ...2001 年 129 巻 3 号 p. 469-475
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーRad51 protein is a eukaryotic homologue of RecA protein that is essential for homologous recombination. We developed a simple procedure for purifying yeast Rad51 protein, characterized its interaction with DNA, and compared it with those of RecA from Escherichia coli and Rad51 from higher eukaryotes. Fractionation of crude extract with 0.2% polyethylenimine eliminated contaminant proteins and nucleic acids, which can perturb the subsequent purification steps. Binding of Rad51 to single-stranded DNA was detected in solution by measuring the fluorescence anisotropy of a fluorescein probe attached to the 5' end of the oligonucleotides. The interaction was stabilized by ATP, as is that of RecA, but was neither stabilized by a non-hydrolysable analog of ATP, nor destabilized by ADP, unlike the interaction of RecA. This character was very similar to that of Xenopus XRad51.1, although the binding of yeast Rad51 to DNA was more sensitive to Mg2+ ion in both the presence and absence of ATP, and was optimal at 5-10mM Mg2+. The dissociation of Rad51 protein from DNA is not, therefore, favored by the hydrolysis of ATP to ADP, in contrast to that of RecA. On the other hand, the high DNA-binding state of the Rad51-DNA complex promoted by ATP appeared to be short-lived. These features may be linked to the lower activity of Rad51 and the fact that Rad51 activity does not require the hydrolysis of ATP.抄録全体を表示PDF形式でダウンロード (1108K) -
Masako Yasugi, Momoyo Amino, Toshiharu Suzuki, Tairo Oshima, Akihiko Y ...2001 年 129 巻 3 号 p. 477-484
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーWe have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus. Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: kcat improved (improved in kcat) and Km-improved (improved in kcat/Km) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH. The ΔGm values for Michaelis intermediate formation of the kcat-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The ΔGm values of Km-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH. Fitting of NAD+ binding was improved in the Km-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in kcat-improved-type, and destabilization of the rate-limiting step in Km-improved type mutants. Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.抄録全体を表示PDF形式でダウンロード (2010K) -
Takeshi Nishikawa, Ayako Kobayashi, Shunji Natori2001 年 129 巻 3 号 p. 485-490
発行日: 2001年
公開日: 2008/11/18
ジャーナル フリーPreviously, we purified the cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP) from Sarcophaga and suggested its participation in the translational regulation of cathepsin B mRNA in this insect. In this study, we isolated a full length cDNA for CBBP. CBBP was an RNA-binding protein that contained four RGG boxes and four zinc finger motifs required for RNA binding. CBBP was shown to be localized in both the nuclei and cytoplasm of Sarcophaga hemocytes. Recombinant CBBP bound to the entire region of cathepsin B mRNA and repressed its translation in vitro.抄録全体を表示PDF形式でダウンロード (2384K)
- |<
- <
- 1
- >
- >|