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Volume 130 (2001)
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Volume 130, Issue 5
Displaying 1-20 of 20 articles from this issue
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Norihisa Fujita, Masanori Yoshida2001 Volume 130 Issue 5 Pages 575-580
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEclosion hormone is an insect neuropeptide that consists of 62 amino acid residues including three disulfide bonds. We have previously reported its hypothetical 3D structure consisting mainly of three α-helices. In this paper, we report the effects of chaper-one proteins on the refolding of denatured eclosion hormone in a redox buffer containing reduced and oxidized glutathione. Urea-denatured eclosion hormone was spontaneously reactivated within 1 min with a yield of more than 90%, while β -mercap-toethanol-denatured eclosion hormone was reactivated in a few minutes with a yield of 75%. Under the same experimental conditions, eclosion hormone treated withβ -mercap-toethanol and urea was reactivated slowly with a yield of 47% over a period of 2h. Protein disulfide isomerase, a eucaryotic chaperone protein, markedly increased the re-activation yield and rate of the totally denatured hormone. GroE oligomers slightly improved the reactivation yield but peptidyl prolyl isomerase had no influence on yield or rate. We propose that the folding pathway of eclosion hormone involves at least two rate-limiting steps, and that protein disulfide isomerase is likely to be involved in the folding in insect neuronal cells.View full abstractDownload PDF (519K) -
Nobuhiro Takahashi, Noboru Sasagawa, Fusako Usuki, Yoshihiro Kino, Hir ...2001 Volume 130 Issue 5 Pages 581-587
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. Patients have a large CTG repeat expansion in the 3' untranslated region of the DMPK gene, which encodes DM protein kinase. RNA trans-dominant models, which hypothe-size that the expanded CUG trinucleotide repeat on DMPK mRNA sequesters a factor or disrupts the RNA metabolism of the DMPK mRNA itself and other mRNAs in a trans dominant manner, have been proposed. A candidate for the sequestered factor, termed CUG-binding protein (CUG-BP), exists in several alternatively spliced isoforms. We found a human isoform with a twelve base insertion (deduced amino acids Leu-Tyr-Leu-Gln) and an isoform with a three base insertion (deduced amino acid Ala) insertion. In order to elucidate the effects of CUG-BP on DMPK expression, we introduced CUG-BP and DMPK cDNA transiently into COS-7 cells. Cotransfection of CUG-BP did not signifi-cantly affect the expression of either wild type or mutant DMPK at the mRNA level. On the other hand, cotransfection of CUG-BP significantly affected the expression of both the wild type and mutant DMPKs at the protein level. This reduction was remarkable when the mutant DMPK construct was used.View full abstractDownload PDF (1714K) -
Hisahiro Tonozuka, Jinqing Wang, Kazuhiro Mitsui, Toshiki Saito, Yoshi ...2001 Volume 130 Issue 5 Pages 589-595
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe protein level of the GTS1 gene product (Gts1p) fluctuated during the oscillation of energy metabolism in continuous culture of the yeast Saccharomyees eerevisiae. Here, we found that the GTS1 mRNA level oscillated with the same periodicity as the metabolic oscillation, suggesting that the expression of GTS1 was regulated at the transcriptional level. As the 5'-upstream sequence of GTS1 contains two short open-reading frames at -310 and -829 by from the initiation codon, we determined the GTS1 promoter required for the oscillatory expression. The upstream sequence was truncated into fragments of 183, 355, 1, 042, and 1, 572 bp, named GTS1pr. 183 and so on, and their effects on the expression of laeZ as a reporter gene and the GTS1 gene itself were examined. The β-galactosidase activity and Gts1p level oscillated in the continuous cultures when genes were expressed under the control of GTS1pr. 183 but not GTS1pr. 355. The disappearance of the metabolic and cell-cycle oscillations in the GTS1-deleted mutant was rescued by the transformation with GTS1pr. 183-GTS1 but not with GTS1pr. 355-GTS1. However, the stress-resistance oscillations were not found in the cells transformed with GTS1pr. 183-GTS1, differing from the case of GTS1pr. 1042-GTS1 reported previously [Wang et al. (2001) FEBS Lett. 489, 81-86]. Thus, we suggest that the 183-bp upstream sequence of GTS1 is basically required for the metabolic oscillation, while the 1, 042-bp upstream sequence is required for oscillations of stress resistance.View full abstractDownload PDF (2301K) -
Takeo Yamaguchi, Juri Miyamoto, Shigeyuki Terada2001 Volume 130 Issue 5 Pages 597-603
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen human erythrocytes were preincubated at 37-52°C under atmospheric pressure before exposure to a pressure of 200 MPa at 37°C, the value of hemolysis was constant (about 43%) up to 45°C but became minimal at 49°C. The results from anti-spectrin anti-body-entrapped red ghosts, spectrin-free vesicles, and N-(1-pyrenyl)iodoacetamidelabeled ghosts suggest that the denaturation of spectrin is associated with such behavior of hemolysis at 49°C. The vesicles released at 200 MPa by 49°C-preincubated erythrocytes were smaller than those released by the treatment at 49°C or 200 MPa alone. The size of vesicles released at 200 MPa was independent of preincubation temperature up to 45°C, and the vesicles released from 49°C-preincubated erythrocytes became smaller with increasing pressure up to 200 MPa. Thus, hemolysis and vesiculation under high pressure are greatly affected by the conformation of spectrin before compression. Since spectrin remains intact up to 45°C, the compression of erythrocytes at 200 MPa induces structural changes of spectrin followed by the release of large vesicles and hemolysis. On the other hand, in erythrocytes that are undergoing vesiculation due to spectrin denaturation at 49°C, compression produces smaller vesicles, so that the hemolysis is suppressed.View full abstractDownload PDF (1521K) -
Kazuhiro Nakagawa, Hajime Masumoto, Hiroyuki Sorimachi, Koichi Suzuki2001 Volume 130 Issue 5 Pages 605-611
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalpain is a heterodimeric, intracellular Ca2+-dependent, “bio-modulator” that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-terminus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into subunits is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca2+ has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (Δ19), but not those lacking only nine residues (Δ9), dissociated into subunits even in the absence of Ca2+. Moreover, both . Δ9 and Δ19 mutants showed an equivalent reduced Ca2+ requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.View full abstractDownload PDF (3022K) -
Maki Onda, Hiroko Hayashi, Tomoyoshi Mita2001 Volume 130 Issue 5 Pages 613-620
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDepartmod at Environmentol Sciences Facility of Science, Osaka Women's University The effect of the interaction of gramicidin (GA) with lysophosphatidylcholine (LPC) on the change in lipid structure upon heat incubation was revealed by differential scanning calorimetry (DSC) and fluorescence spectroscopy. DSC showed a large endothermic transition in both pure LPC micelles and GA-containing LPC micelles after prolonged heat incubation at 70°C. To elucidate this behavior, fluorescence spectra of 1- anilinonaphthalene-8-sulfonate embedded in LPC micelles were measured. About 40% of the resultant LPC micelles was found to be transformed into the interdigitated gel structures after prolonged heat incubation. On the other hand, intrinsic fluorescence spectra of GA-containing LPC micelles caused a blue-shift of the emission maxima with incubation time, suggesting that tryptophans near the C-terminus of GA moved into a more apolar environment. In addition, GA-containing LPC micelles caused quenching of fluorescence with incubation time, due to the interaction between GA molecules. To determine the location of GA in LPC membranes, surface pressure was measured using the mixed monolayers composed of GA and LPC. The result suggests that GA molecule is localized by interdigitating the C-terminal part of adjacent to acyl chain of LPC.View full abstractDownload PDF (705K) -
Tomoyuki Numata, Makoto Kimura2001 Volume 130 Issue 5 Pages 621-626
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al (2000) Biochein. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3', 5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased Km, and 27.6-fold decreased kcat, while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the kcat, value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (kcat/Km) with a minimum Km value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2', 3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner.View full abstractDownload PDF (1114K) -
Lynn Farh, Shao-Ying Hwang, Lary Steinrauf, Hucy-Jenn Chiang, David Sh ...2001 Volume 130 Issue 5 Pages 627-635
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an ironsulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe2+ content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.View full abstractDownload PDF (3139K) -
Yasuzo Nishina, Kyosuke Sato, Ruiwen Shi, Chiaki Setoyama, Retsu Miura ...2001 Volume 130 Issue 5 Pages 637-647
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the structural modulation of ligands and their interaction in the active-site nanospace when they form charge-transfer (CT) complexes with D-amino acid oxidase (DAO) in three redox states, we compared Raman bands of the ligands in complex with DAO with those of ligands free in solution. Isotope-labeled ligands were synthe-sized for assignments of observed bands. The COO-stretching of ligands observed around, 1, 370 cm-1 downshifted by about 17 cm-1 upon complexation with oxidized, semi-quinoid and reduced DAO, except for the case of reduced DAO-N-methylisonicotinate complex (8 cm-1 downward shift); the interaction mode of the carboxylate group with the guanidino group of Arg283 and the hydroxy moiety of Tyr228 of DAO is similar in the three redox states. The C=N stretching mode (1, 704cm-1) of Δ1-piperideine-2-carboxy-late (D1PC) downshifted to 1, 675 and 1, 681 cm-1 upon complexation with reduced and semiquinoid DAO, respectively. The downward shifts indicate that the C=N bond is weakened upon the complexation. This is probably due mainly to charge-transfer (CT) interaction between D1PC and semiquinoid or reduced flavin, i. e., the partial electron donation from the highest occupied molecular orbital (HOMO) of reduced flavin or a singly occupied molecular orbital (SOMO) of semiquinoid flavin to the lowest unoccupied molecular orbital (LIMO), an antibonding orbital, of D1PC. This speculation was supported by the finding that the magnitude of the shift is smaller by 5cm-1 (observed at 1, 680cm-1) in the case of reduced DAO reconstituted with 7, 8-Cl2-FAD, whose reduced form has lower electron-donating ability than natural reduced FAD. The amount of electron flow was estimated by applying the theory of Friedrich and Person [(1966) J. Chem. Phys. 44, 2166-2170] to these complexes; the amounts of charge transfer from reduced FAD and reduced 7, 8-Cl2-FAD to D1PC were estimated to be about 10 and 8% of one electron, respectively, in the CT complexes of reduced DAO with D1PC.View full abstractDownload PDF (1972K) -
Takeo Imai, Katsuhiko Taguchi, Yoko Ogawara, Daijiro Ohmori, Fumiyuki ...2001 Volume 130 Issue 5 Pages 649-655
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe_ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid se-quence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100°C, while the half-life of the aerobically-purified ferredoxin is 10h at 100°C. Both the anaerobically-and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.View full abstractDownload PDF (2097K) -
Yoshiyuki Mizushina, Nobuyuki Kasai, Fumio Sugawara, Akira Iida, Hirom ...2001 Volume 130 Issue 5 Pages 657-664
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase β (pol β), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol β and topo II. Here, we report the molecular interaction of LCA with pol β and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol β(Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol β and topo II are three-dimensionally very similar.View full abstractDownload PDF (2748K) -
Yasuyuki Imai, Yasumi Matsuura, Yousuke Ono, Tomoyuki Ishikawa, Yukish ...2001 Volume 130 Issue 5 Pages 665-670
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cytotoxic effects of Shiga-like toxins from enterohemorrhagic Escherichia coli O157:H7 depend on the recognition of carbohydrate determinants by B subunits. As a specific carbohydrate ligand, globotriaosylceramide has been characterized. We developed an alternative binding assay using multivalent carbohydrate ligands. We prepared globotriose-conjugated poly-lysine, and measured their binding to immobilized recombinant B subunits by an ELISA format. The signals representing ligand binding were dependent on the amount of immobilized B subunits as well as on the concentration of the ligands. The ligand binding activity was lost in an acidic environment, in which changes in the local conformation of the B subunits have been reported. Furthermore, pH dependent dissociation of the ligands from the B subunits was observed. We also demonstrate that antiserum from mice immunized with the B subunits specifically interferes with ligand binding. This suggests further potential for an assay to screen for blocking antibodies that could inhibit toxin internalization into host cells.View full abstractDownload PDF (453K) -
S. Ghobadi, S. Safarian, A. A. Moosavi-Movahedi, A. B. Ranjbar2001 Volume 130 Issue 5 Pages 671-677
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction between n-octyl-β-D-glucopyranoside (octyl glucoside) and bovine liver glutamate dehydrogenase (GDH) was studied using techniques including equilibrium dialysis, UV-spectrophotometry, circular dichroism (CD), fluorescence energy transfer and extrinsic spectrofluorometry in 50mM sodium phosphate buffer solution (pH 7.6). The equilibrium dialysis experiment showed a higher binding of octy1 glucoside to GDH that induces up to 80% enzyme inhibition in 20mM octy1 glucoside solution. The CD study indicated that GDH retains its secondary structure in the presence of octyl glucoside, but loses a degree of its tertiary structure by acquiring a more extended tertiary structure. Measurement of the binding of a hydrophobic fluorescent probe, 1-anilinonaphthalene-8-sulfonate (ANS), to GDH revealed that the binding of ANS to GDH is increased in the presence of octyl glucoside, a finding that may be interpreted in terms of the increment of sin-face hydrophobic patch(es) of GDH because of its binding to octyl glucoside. Fluorescence energy transfer studies also showed more binding of the reduced coenzyme (NADH) to GDH and the Lineweaver-Burk plots (with respect to NADH) indicate the existence of substrate inhibition in the resence of octyl glucoside. These observations are aimed at explaining the formation of the molten globule-like structure of GDH, which is induced by a non-ionic detergent such as octyl glucoside.View full abstractDownload PDF (2471K) -
Hugo Tremblay, Tsugihisa Yamaguchi, Tamo Fukamizo, Ryszard Brzezinski2001 Volume 130 Issue 5 Pages 679-686
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)6] by site-directed mutagenesis, thermal unfolding, and (GlcN)6 digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)6 complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (Tm), usually observed upon the addition of (GlcN)n to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)6] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.View full abstractDownload PDF (4272K) -
Naoki Ichikawa, Ayako Karaki, Miho Kawabata, Saori Ushida, Mika Mizush ...2001 Volume 130 Issue 5 Pages 687-693
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial ATP synthase (F1 F0-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement. A total of 22 mutants were isolated and characterized. Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein. The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity. Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH. The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable. The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity. Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F1 F0-ATPase. These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibi-tion of F1, rather than binding to the enzyme.View full abstractDownload PDF (1026K) -
The Structure of the Archaebacterial Ribosomal Protein S7 and Its Possible Interaction with 16S rRNAHartuni Hosaka, Min Yao, Makoto Kimura, Isao Tanaka2001 Volume 130 Issue 5 Pages 695-701
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibosomal protein S7 is one of the ubiquitous components of the small subunit of the ribosome. It is a 16S rRNA-binding protein positioned close to the exit of the tRNA, and it plays a role in initiating assembly of the head of the 30S subunit. Previous structural analyses of eubacterial S7 have shown that it has a stable α-helix core and a flexible β-arm. Unlike these eubacterial proteins, archaebacterial or eukaryotic S7 has an N-terminal extension of approximately 60 residues. The crystal structure of S7 from archaebacterium Pyrococcus horikoshii (PhoS7) has been determined at 2.1 Åresolution. The final model of PhoS7 consists of six major α-helices, a short 310-helix and two β-stands. The major part (residues 18-45) of the N-terminal extension of PhoS7 reinforces the α-helical core by well-extended hydrophobic interactions, while the other part (residues 46-63) is not visible in the crystal and is possibly fixed only by interacting with 16S rRNA. These differences in the N-terminal extension as well as in the insertion (between αl and α2) of the archaebacterial S7 structure from eubacterial S7 are such that they do not necessitate a major change in the structure of the currently available eubacterial 16S rRNA. Some of the inserted chains might pass through gaps formed by helices of the 16S rRNA.View full abstractDownload PDF (2643K) -
Hitoshi Aoshima, Sheikh Julfikar Hossain, Koutaro Hamamoto, Terufumi Y ...2001 Volume 130 Issue 5 Pages 703-709
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the kinetics of both the potentiation and desensitization of the response of ionotropic GABA receptors (GABAA receptors) in the presence of various compounds, we expressed receptors composed of α1 and β1subunits by injectingcells with the cRNAs synthesized from cloned bovine GABAA receptor cDNAs and measured the elec-trical responses of the cells electrophysiologically with or without the compounds. The potentiation of the GABAA receptor-mediated response was quantitatively analyzed using a simple model with the assumption that thereceptors have two identical binding sites for GABA molecules with a dissociation constant of Kp, and one potentiation site for the compound with a dissociation constant of Kp, and that the binding of the compound to the potentiation site only increases the affinity of the GABA binding sites, changing K1 to K1p.The estimated Kp and K1p were dependent on the functional groups and the chainlength of the compounds. These results could be satisfactorily analyzed using this simple model. The potentiation of the GABAA receptor-mediated response by the components of essential oils used for aromatherapy was also examined. These com-pounds accelerated the decay of the response, possibly due to desensitizationof the receptors, which was also analyzed on the basis of the model.View full abstractDownload PDF (1887K) -
Takiko Gouda, Masayuki Kuroda, Toshiaki Hiramatsu, Kaori Nozaki, Teruo ...2001 Volume 130 Issue 5 Pages 711-717
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe cloned a gene which enabled Escherichia coli mutant host cells lacking all ofthe major Na+/H+ antiporters to grow in the presence of 0. 2 M NaCl from chromosomal DNA of Bacillus subtilis ATCC9372. An Na+/H+ antiport activity was observedwith membrane vesicles prepared from E coli cells possessing the cloned gene, but not with vesicles from the host cells. Lithium ion was also a substrate for the antiporter. We sequenced the cloned DNA and found one open reading frame (designated nhaG) preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence. The deduced amino acid sequence of NhaG suggested that it consisted of 524 residues and that the calculated molecular mass was 58. 1 kDa. None of the bacterial Na+/H+ antiporters so far reported, except NhaP of Pseudomonas aeruginosa and SynN-haP (NhaSl) of Synechocystis sp., showed significant sequence similarity with the NhaG. However, the NhaP, the SynNhaP, animal NHEs (Na+/H+ exchangers), and some hypothet-ical Na+/H+ antiportersof several organisms showed significant sequence similarities with the NhaG. Interestingly, the entire DNA region corresponding to the nhaG gene is missing in the reported complete genome sequence of B. subtilis strain 168. We detected a band that hybridized with the nhaG DNA in chromosomal DNA from B. subtilis ATCC9372 but not with that from strain 168. The missing DNA region (1, 774 base pairs) is sandwiched by two identical sequences, TTTTCTT.View full abstractDownload PDF (1315K) -
2001 Volume 130 Issue 5 Pages 719a
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (22K) -
2001 Volume 130 Issue 5 Pages 719b
Published: 2001
Released on J-STAGE: November 18, 2008
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