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Volume 128 (2000)
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Volume 128, Issue 2
Displaying 1-22 of 22 articles from this issue
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Tomoko Kadowaki, Koji Nakayama, Kuniaki Okamoto, Naoko Abe, Atsuyo Bab ...2000 Volume 128 Issue 2 Pages 153-159
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPorphvromonas gingivalis, one of the major causative agents of periodontal diseases, produces large amounts of arginine- and lysine-specific cysteine proteinases in cell-asso-ciated and secretory forms, which are now referred to as Arg-gingipain (Rgp) and Lysgingipain (Kgp), respectively. A number of studies have revealed that these proteinases are closely associated with the periodontopathogenesis of this bacterium: destruction of periodontal connective tissues, disruption of host defense mechanisms, and development and maintenance of inflammation in periodontal pockets. With respect to the physiology of the bacterium, Rgp and Kgp are indispensable for it to obtain nutrients from the environment, since it cannot utilize saccharides as carbon/energy sources for growth and totally depends on peptides and amino acids that are provided from environmental proteins by Rgp and Kgp. Furthermore, proteolytic activities of Rgp and Kgp contribute to processing/maturation of various cell-surface proteins of P. gingiaalis, such as fimA fimbrilin (a subunit of major fimbriae), 75-kDa protein (a subunit of minor fimbriae), hemagglutinins, and the hemoglobin receptor protein, which are important for the bacterium to colonize and proliferate in the gingival crevice and to invade the periodontium. These findings strongly indicate critical roles of Rgp and Kgp in the Virulence of P. gingiaalis.View full abstractDownload PDF (2272K) -
Yoshiaki Kouzuma, Hideko Inanaga, Keiko Doi-Kawano, Nobuyuki Yamasaki, ...2000 Volume 128 Issue 2 Pages 161-166
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sea cDNA fragment as a hybridization probe. The cDNA insert comprises 1, 093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sea. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.View full abstractDownload PDF (1827K) -
Satoshi Fujimura, Kiyotaka Oshikawa, Shigeyuki Terada, Eiji Kimoto2000 Volume 128 Issue 2 Pages 167-173
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of brevilysin H6 (H6), a zinc-protease isolated from Gloydius halys brevicaudus venom, was determined by a manual Edman degradation method. H6 has an amino-terminal pyroglutamic acid and consists of a total of 419 residues. An N-linked sugar chain is attached at Asn-181. The molecule is composed of three domains (metalloprotease, disintegrin-like and cysteine-rich domains), as commonly found in other high molecular mass metalloproteases from snake venoms. In the absence of calcium ions, H6 is autocatalytically degraded with a half-life of 47 min to give 29 and 45 kDa fragments, which correspond to residues 208-419 and 99-419 of H6, respectively. Thus, the autoproteolysis seemed to start from the cleavage of either the Leu98-Leu99 or Asp207-IIe208 bond. Calcium ions suppressed both the formation of the 45 kDa fragment and the rate of autoproteolysis. Calcium ions also contributed to the stability of H6 against pH, heating, urea and cysteine. More than twenty-five peptide bonds adjacent to hydrophobic residues in the metalloprotease domain were progressively cleaved during the autoproteolysis.View full abstractDownload PDF (1691K) -
Yasushi Makino, Kaoru Omichi, Nahoki Kuraya, Hideyuki Ogawa, Hitoshi N ...2000 Volume 128 Issue 2 Pages 175-180
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structures of N-glycans of human blood clotting factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.View full abstractDownload PDF (911K) -
Yoshio Imai, Isamu Matsunaga, Emi Kusunose, Kosuke Ichihara2000 Volume 128 Issue 2 Pages 189-194
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFatty acid α-hydroxylase from Sphingomonas paucimobilis is a hydrogen peroxide-de-pendent cytochrome P450 (P450) enzyme (P450SPa). In this study, heme-ligand exchange reactions of P450SPa were investigated using the optical spectroscopic method and compared with those of various P450s. Alkylamines (C_??_5) induced changes in the spectrum of ferric P450SPa to one typical of a nitrogenous ligand-bound low-spin form of ferric P450, although their affinities were lower than those for other P450s, and a substrate, laurate, did not interfere with the binding in contrast with in the cases of other P450s. Other compounds having a nitrogen donor atom to the heme iron of P450, including pyridine or 1-methylimidazole, induced no change in the spectrum of P450SPa in either the ferric or ferrous state. Practically no spectral change was observed on the addition of alkyl isocyanides to ferric P450s. On the other hand, cyanide induced a change in the spectrum of ferric P450SPa to one characteristic of cyanide-bound form of ferric P450. The affinity of cyanide increased when the substrate was added, in contrast with in the cases of other P450s. Ferrous P450SPa combined with CO and alkyl isocyanides, and the affinity for CO was of the same order of magnitude as in the cases of other P450s. These findings suggest a unique heme environment of P450SPa, in which most compounds usually acting as external ligands of ferric P450s are prevented from gaining access to the heme iron of P450SPa. The unique properties of the hydroxylase reaction catalyzed by P450SPa, where an oxygen atom of hydrogen peroxide but not of molecular oxygen is utilized and incorporated into a fatty acid at its a position, is possibly related with such a specific heme environment of this P450. A possible mechanism for the peroxygenase reaction of P450SPa is proposed.View full abstractDownload PDF (2778K) -
Tetsuhiro Kawabe, Erika Fujihira, Akihito Yamaguchi2000 Volume 128 Issue 2 Pages 195-200
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC. The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium. In this study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed. Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody. After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 KDa band, indicating that AcrA co-precipitated with AcrB. Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with [14C] N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence.View full abstractDownload PDF (2584K) -
Sadao Wakabayashi, Hidemasa Yoshida, Toshio Shigekiyo, Takehiko Koide2000 Volume 128 Issue 2 Pages 201-206
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe reported the first case of a congenital histidine-rich glycoprotein deficiency (HRG Tokushima) in which substitution of Gly85 with Glu (G85E) in the first cystatin domain resulted in intracellular degradation and a low plasma level of HRG [Shigekiyo, T. et at. (1998) Blood 91, 128-133]. Recently, we identified the gene mutation of a second case of HRG deficiency as a Cys223 to Arg (C223R) mutation in the second eystatin domain. To investigate the molecular and cellular bases of these deficiencies, we expressed these HRG mutants in baby hamster kidney (BHK) cells. Pulse-chase experiments in the absence and presence of various proteinase inhibitors revealed that, while wild-type HRG was completely secreted during 4-h chase periods, both the G85E and C223R mutants were only partially secreted and primarily degraded within the cells. The intracellular degradation of the C223R mutant was almost completely inhibited in the presence of a proteasome inhibitor, lactacystin, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, resulting in increased secretion of the C223R mutant, and thus implicating the proteasome system in this degradation process. In contrast, the sum of the amounts of the G85E mutant inside and outside the cells decreased during the chase periods even in the presence of the proteasome inhibitor, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, although proteasome-specific inhibitor lactacystin and one of the cysteine protease inhibitors, E-64-d, prevented the intracellular degradation. These results suggested that intracellular degradation of G85E HRG occurred to some extent through a hitherto unknown mechanism. Similar studies involving recombinant mutants in which Gly85 or Cys223 was replaced with several other amino acids revealed that proteins with mutations leading to the destruction of the predicted β-sheet structure of the cystatin domains were eliminated by the intracellular quality control system.View full abstractDownload PDF (564K) -
Hua-Shan Huang, Kiyoshi Ito, Tsutomu Kabashima, Tadashi Yoshimoto2000 Volume 128 Issue 2 Pages 207-211
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene glpK, encoding glycerol kinase (GIpK) of Thermus aquaticus, has recently been identified. The protein encoded by glpK was found to have an unusually high identity of 81% with the sequence of GlpK from Bacillus subtilis. Three residues (Arg-82, Glu-83, and Asp-244) of T. aquaticus GlpK are conserved in all the known GlpK sequences, including those from various bacteria, yeast and human. The roles that these three residues play in the catalytic mechanism were investigated by using site-directed mutagenesis to produce three mutants: Arg-82-Ala, Glu-83-Ala, and Asp-244-Ala. Replacement of Asp-244 by Ala resulted in a complete loss of activity, thus suggesting that Asp-244 is important for catalysis. Taking kcat/Km as a simple measure of catalytic efficiency, the mutants Arg-82-Ala and Glu-83-Ala were judged to cause 190- and 37, 000-fold decrease, respectively, when compared to the wild-type GlpK. Thus, these three residues play a critical role in the catalytic mechanism. However, only mutant Glu-83-Ala was cleaved by α-chymotrypsin, and proteolysis studies showed that the mutant Glu-83-Ala involves a change in the exposure of Tyr-331 at the α-chymotrypsin site. This indicates a large domain conformational change, since the residues corresponding to Glu-83 and Tyr-331 in the Escherichia coli GlpK sequence are located in domain IB and domain IIB, respectively. The apparent conformational change caused by replacement of Glu-83 leads us to propose that Glu-83 is an important residue for stabilization of domain conformation.View full abstractDownload PDF (2732K) -
Yasuzo Nishina, Kyosuke Sato, Retsu Miura, Kiyoshi Shiga2000 Volume 128 Issue 2 Pages 213-223
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe investigated the mechanism of recognition and activation of substrate by D-amino acid oxidase (DAO) by thermodynamical and spectrophotometric methods using zwitterionic figands [N-methylisonicotinate (NMIN), trigonelline, and homarine] and monoanionic ligands as model compounds of the substrate and the product. In terms of the charge within the substrate D-amino acid, monoanionic (e. g., benzoate), zwitterionic (e. g., NMIN), and dianionic (e. g., terephthalate) ligands are thought to be good models for neutral, basic, and acidic amino acids, respectively, because when a substrate binds to DAO, as previously reported, the α-anunonium group (-NH3+) probably loses a proton to become neutral (-NH2) before the oxidation. Zwitterionic ligands can also be good model compounds of product in the purple complex (the complex of reduced DAO with the product imino acid), because the imino nitrogen of the imino acid is in a protonated cationic form. We also discuss electrostatic interaction, steric effect, and charge-transfer interaction as factors which affect the affinity of substrate/ligand for DAO. Monoanionic ligands have high affinity for neutral forms of oxidized and semiquinoid DAO, while zwitterionic ligands have high affinity for anionic forms of oxidized, semiquinoid, and reduced DAO; this difference was explained by the electrostatic interaction in the active site. The low affinity of homarine (N-methylpicolinate) for oxidized DAO, as in the case of O-methylbenzoate, is due to steric hindrance: one of the ortho carbons of benzoate is near the phenol carbons of Tyr228 and the other ortho carbon is near the carbonyl oxygen of Gly313. The correlation of the affinity of meta- and para -substituted benzoates for oxidized DAO with their Hammet's O values are explained by the HOMO-LUMO interaction between the phenol group of Tyr224 and the benzene ring of benzoate derivative. The pKa of neutral flavin [N (3) -H of oxidized flavin, N (5) -H of semiquinoid flavin, and N (1) -H of reduced flavin] decreases by its binding to the apoenzyme. The magnitude of the decrement is oxidized flavin semiquinoid flavin < reduced flavin. The largest factor in the substantially low pKa of reduced flavin in DAO is probably the steric hindrance between the hydrogen atom of H-N (1) (flavin) and the hydrogen atom of H-N of Gly315, which becomes significant when a hydrogen is bound to N(1) of flavin.View full abstractDownload PDF (3457K) -
Ayami Matsushima, Tsugumi Fujita, Takeru Nose, Yasuyuki Shimohigashi2000 Volume 128 Issue 2 Pages 225-232
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the ligand/receptor interaction, the side chain phenyl group of phenylalanine (Phe) is involved in a so-called hydrophobic interaction, in which the Phe-phenyl group func-tions as a π element or merely as a hydrophobic element. The thrombin receptor-teth-ered ligand SFLLRNP consists of the Phe-2 residue essential for receptor activation. In order to explore the molecular characteristics of this Phe-2-phenyl group, a complete set of S/Phe/LLRNP peptides comprising six different difluorophenylalanine isomers [(F2)Phe] was newly synthesized and assayed to evaluate their ability to induce the aggregation of human platelets. The assay results clarified several important structural elements to conclude that Phe-2-phenyl of S/Phe/LLRNP is in the edge-to-face CH/π interaction with the receptor aromatic group, utilizing the Phe-phenyl edge along with adjacent benzene hydrogens at positions (2-3) or (5-6). It was also found that the fluo-rine atom at position 4 increases the acidity of the hydrogen mainly at its ortho position, resulting in a reinforcement of the CH/π interaction and thus in an enhancement of biological activity. The H→F replacement in the benzene ring was found to provide an effective structural examination to the Phe residue; i.e., to identify the hydrogens in the CH/π interaction, and to strengthen the CH/π interaction.View full abstractDownload PDF (1210K) -
Amit Choudhury, Nicolas Moniaux, John P. Winpenny, Michael A. Hollings ...2000 Volume 128 Issue 2 Pages 233-243
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe human MUC4 gene is not expressed in normal pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors. To investigate the tumor-asso-ciated expression, MUC4 eDNA was cloned from a human pancreatic tumor cell line cDNA expression library using a polyclonal antibody raised against human deglycosy-lated mucin and RT-PCR. Pancreatic MUC4 cDNA shows differences in 12 amino acid residues in the non-tandem repeat coding region with no structural rearrangement as compared with tracheal MUC4. The full-length MUC4 cDNA includes a leader sequence, a serine and threonine rich non-tandem repeat region, a central large tandem repeat domain containing 48 bp repetitive units, regions rich in potential N-glycosylation sites, two cysteine-rich domains, EGF-like domains, and a transmembrane domain. We also report the presence of a new EGF-like domain in MUC4 eDNA, located in the cysteine-rich region upstream from the first EGF-like domain. Four distinct splice events were identified in the region downstream of the central tandem repeat domain that generate three new MUC4 eDNA sequences (sv4, sv9, and sv10). The deduced amino acid sequences of two of these variants lack the transmembrane domain. Furthermore, two unique forms of MUC4 (MUC4/Y and MUC4/X) generated as a result of alternative splicing lack the salient feature of mucins, the tandem repeat domain. A high degree of poly-morphism in the central tandem repeat region of MUC4 was observed in various pancreatic adenocarcinoma cell lines, with allele sizes ranging from 23.5 to 10.0 kb. MUC4 mRNA expression was higher in differentiated cell lines, with no detectable expression in poorly differentiated pancreatic tumor cell lines.View full abstractDownload PDF (3079K) -
Junichiro Futami, Hiroko Tada, Masaharu Seno, Shinya Ishikami, Hidenor ...2000 Volume 128 Issue 2 Pages 245-250
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to stabilize human RNase 1 by introduction of an intramolecular cross-link, a mutant protein (4-118CL RNase 1), in which Arg4 and Val118 are replaced with cysteine residues and linked by a disulfide bond, was designed and expressed in Escherichia coli as inclusion bodies. The 4-118CL RNase 1 that refolded under redox conditions was a monomer without free SH groups and retained 11% of the activity of the wild-type recombinant RNase 1, indicating that the mutant enzyme was correctly folded with the formation of an additional disulfide bond between Cys4 and Cys118. From guanidium chloride denaturation experiments based on the assumption of a two-state transition for unfolding, it was demonstrated that the introduction of the present cross-link increased the thermodynamic stability of RNase 1 by 2.0 kcal/mol. This value was lower than that, 5.4 kcal/mol, theoretically calculated from the reduction of chain entropy of the unfolded state due to the introduction of the cross-link. These results suggest that the present cross-link also destabilized the folded state of RNase 1 by 3.4 kcal/mol. Along with the increase in the thermodynamic stability, the stability of RNase 1 against trypsin digestion was also significantly increased by the introduction of this cross-link. It is likely, although not proven, that stabilized human RNases are favorable for clinical use, because human RNase-based immunotoxins should have long half-lives as to proteolytic degradation after endocytosis.View full abstractDownload PDF (1882K) -
Mamoru Ikemoto, Takemistsu Furuchi, Hiroyuki Arai, Keizo Inoue2000 Volume 128 Issue 2 Pages 251-259
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe removal of cholesterol from macrophages is important for reversing foam cell for-mation. In a previous study, we demonstrated that mouse peritoneal macrophages in culture secrete significant amounts of unesterified cholesterol from the lysosomes into the medium during endocytosis and subsequent metabolism of cholesterol-containing liposomes [Furuchi, T., Aikawa, K., Arai, H., and Inoue, K. (1993), J. Biol. Chem. 268, 27345-27348]. In this study, we found that at least two distinct mechanisms are involved in this process. The efflux of unesterified cholesterol into the medium was greatly suppressed by pregnenolone, an inhibitor of lysosomal cholesterol transport, but an appreciable proportion of the unesterified cholesterol was still released into the medium. Analysis of the medium containing the secreted cholesterol by NaBr density gradient ultracentrifugation revealed that the unesterified cholesterol was distributed in two different density peaks (bottom and d=_??_1.1). The d=_??_1.1 peak material formed high-den-sity lipoprotein (HDL)-like particles that were produced and secreted by the macro-phages. The lipid components of these particles were phosphatidylcholine and sphingo-myelin, while the sole protein component was apolipoprotein E (apo E). Treatment with pregnenolone completely abolished the production of these HDL-like particles but had little effect on the bottom fractions. These data indicate that macrophages release lysosomal cholesterol via both pregnenolone-sensitive and -insensitive pathways, and that only the cholesterol secreted through the pregnenolone-sensitive pathway is associated with endogenously synthesized apo E-containing HDL-like particles. Moreover, we found that the pregnenolone-sensitive pathway operated independently of the presence or absence of exogenous HDL, whereas secretion via the pregnenolone-insensitive pathway was greatly stimulated by exogenously added HDL.View full abstractDownload PDF (2617K) -
Tung-Ligan Chung, Lynn Farh, Ya-Lei Chen, David Shiuan2000 Volume 128 Issue 2 Pages 261-269
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe recombinant clone expressing a 60 kDa (P 60) antigen was isolated from Escherichia coli by screening a lambda EMBL3 genomic library using rabbit produced antiserum against Mycoplasma hyopneurnoniae. Sequence analysis revealed that an interrupted (by a UGA codon) open reading frame coding for a 72 kDa protein (P 72) may contain the P 60 antigen gene. Western blot analysis with an anti-P 60 monospecific antibody con-firmed the presence of a P 72 antigen from the total protein of M. hyopneumoniae, and a 72 kDa protein was also expressed in E. coli after changing the codon (UGA to UGG) by site-directed mutagenesis. BLAST (Basic Local Alignment Search Tool) comparison showed that the amino acid sequences of P 72 share approximately 70% homology with the phosphotransferase enzyme I (PTSI) of bacteria and other mycoplasma species. The biological function of the P 72 cytosolic protein was further confirmed by complementation using an E. coli ptsI mutant. The bacterial phosphoenolpyruvate-sugar phospho-transferase system (PTS) is known to mediate the uptake and phosphorylation of carbohydrates and to be involved in signal transduction. The immune responses of specific pathogen free (SPF) pigs and farm animals toward this unique antigen were observed. The transcription start positions of the PTSI gene were determined in M. hyopneumoniae and E. coli by primer extension experiments and the promoter site was also predicted.View full abstractDownload PDF (2305K) -
Kazuaki Ohtsubo, Seiichi Imajo, Masamichi Ishiguro, Takako Nakatani, S ...2000 Volume 128 Issue 2 Pages 283-291
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAll members of a glucuronyltransferase (GlcAT) gene family cloned to date contain four conserved regions (modules I-IV), which are widely located in the catalytic domain. In order to understand the biological significance of these modules, we investigated the structure-function relationship of GlcAT-P by means of the combination of site-directed mutagenesis and computer aided three-dimensional modeling. The wild-type and mutant GlcAT-Ps were expressed in Escherichia coli as glutathione-S-transferase (GST)-fused soluble proteins. Most of the mutants in which a polar amino acid within the modules was replaced with alanine lost their transferase activity almost completely, while all of the mutants in which the replacement was outside these modules retained the original catalytic activity. A three-dimensional (3-D) model of GIcAT-P was constructed by computer simulation with the three-dimensional structure of adenylate kinase (1AKE) as a template. This model predicted that the large catalytic domain of GlcAT-P forms a globular shape with a Rossmann-fold motif consisting of five α-helix and β-sheet repeats. The putative catalytic pocket consisting mainly of modules I-III is surrounded by a cluster of polar amino acids, which are essential for the transferase activity and also for the binding to the acceptor substrate (essential amino acids), asialo-orosomu-coid. There is the second cluster of essential amino acids almost on the opposite surface of the molecule, in which an aspartic acid repeat (DDD) is located. The biological signifi-cance of the second cluster is currently not clear but it may be associated with the inter-action of the enzyme with modulation molecules, manganese and membrane phospholipids.View full abstractDownload PDF (3788K) -
Kenzi Suzuki, Eri Asao, Yasuhiko Nakamura, Mihoko Nakamura, Kuniharu O ...2000 Volume 128 Issue 2 Pages 293-299
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression systems for the sal gene encoding salicylate hydroxylase from Pseudomonas putida S-1 were examined and some constructs were expressed in these systems. By cultivation of Escherichia coli BL 21 (DE3)/pSAH8 in LB medium at 37°C with isopropyl-β-D-thiogalactopyranoside as the inducer, salicylate hydroxylase was overexpressed mainly in the form of inclusion bodies. Lower temperature cultivation at 20°C after induction resulted in a large amount of the enzyme in the soluble form. The E. coli clone harboring the recombinant plasmid produced a 45 kDa protein that appeared to be electrophoreti-cally and immunochemically identical to the P. putida enzyme and contained the same N-terminal amino acid sequence. This recombinant DNA product also exhibited proper-ties characteristic of a flavoprotein and was fully functional as salicylate hydroxylase. Based on chemical modification of the salicylate hydroxylase from P. putida, Lys163 was previously proposed to be the NADH binding site. In this study, to obtain a better under-standing of the predicted role of Lys163, this residue in the active center of salicylate hydroxylase was replaced with Arg, Gly, or Glu by conventional site-directed mutagenesis. Kinetic studies using these mutant enzymes and the recombinant enzyme revealed increases in apparent Km values for NADH in the order of wild-type enzyme>K163R>K163G>K163E, with some decreases in Vmax, Examination of the recombinant enzyme and K163G indicated that the pH dependency of Km on NADH with pKa10.5 is lost by mutation despite the lack of changes in Vmax values, suggesting a requirement for the lysine residue as the NADH binding site. Based on these results, Lys163 is proposed to play a role in the binding of NADH at the active site through an ionic bond rather than playing a role in catalysis.View full abstractDownload PDF (2817K) -
Wakako Suhara, Mitsutoshi Yoneyama, Tomokatsu Iwamura, Shoko Yoshimura ...2000 Volume 128 Issue 2 Pages 301-307
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCellular genes including the type I interferon genes are activated in response to viral infection. We previously reported that IRF-3 (interferon regulatory factor 3) is specifi-cally phosphorylated on serine residues and directly transmits a virus-induced signal from the cytoplasm to the nucleus, and then participates in the primary phase of gene induction. In this study, we analyzed the molecular mechanism of IRF-3 activation further. The formation of a stable homomeric complex of IRF-3 between the specifically phosphorylated IRF-3 molecules occurred. While virus-induced IRF-7 did not bind to p300, the phosphorylated IRF-3 complex formed a stable multimeric complex with p300 (active holocomplex). Competition using a synthetic phosphopeptide corresponding to the activated IRF-3 demonstrated that p300 directly recognizes the structure in the vicinity of the phosphorylated residues of IRF-3. These results indicated that the phos-phorylation of serine residues at positions 385 and 386 is critical for the formation of the holocomplex, presumably through a conformational switch facilitating homodimer for-mation and the generation of the interaction interface with CBP/p300.View full abstractDownload PDF (2381K) -
Kikuo Ogata, Rie Ohno, Ryo Morishita, Yaeta Endo, Akira Wada2000 Volume 128 Issue 2 Pages 309-313
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS(1) Escherichia coli 70 S ribosomes showed intrinsic ATPase and GTPase activities, although they were much lower than those of rat liver ribosomes. The latter activity was higher than the former one. (2) The ATPase activity was inhibited by GTP and GMP-P(NH)P, and the GTPase activity was inhibited by ATP and AMP-P(NH)P, indicating a close relationship between the two enzymes. (3) Elongation components alone or in com-bination enhanced the ATPase activity, indicating the possible correlation of ribosomal ATPase with elongational components. (4) Vanadate at the concentrations that did not inhibit the GTPase activities of EF-Tu and EF-G, depressed the poly (U)-dependent polyphe synthesis, suggesting that ribosomal ATPase (GTPase) participates in peptide elongation by inducing positive conformational changes of ribosomes required for the attachment of elongational components.View full abstractDownload PDF (381K) -
Koichi Inano, Isao Suetake, Takayuki Ueda, Yasuko Miyake, Masahiko Nak ...2000 Volume 128 Issue 2 Pages 315-321
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMaintenance-type DNA methyltransferase (Dnmt1) is usually down-regulated in nonproliferating cells. In the present study, we detected significant expression of Dnmt1 protein in adult mouse brain where the majority of the cells are in a post-mitotic state. A significant amount of Dnmt1 protein was fractionated into the post-nuclear fraction for both cerebrum and cerebellum. The Dnmt1 in this fraction was enzymatically active. An immunofluorescence study revealed that Dnmt1 protein was mainly expressed in neurons and seemed to be localized in the cytoplasmic compartment. Primary culturing of neurons confirmed the expression and localization of Dnmt1 in the cytoplasmic compartment. The findings that the Dnmtl transcript in the brain utilized the somatic-type exon and that the apparent size of the Dnmt1 protein in the cytoplasm was identical to that in proliferating culture cells indicate that the cytoplasmic Dnmt1 in neurons was of the somatic-type.View full abstractDownload PDF (2650K) -
Byung-Taek Kim, Wan-Seok Kim, Yeong Shik Kim, Robert J. Linhardt, Dong ...2000 Volume 128 Issue 2 Pages 323-328
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel type of heparinase (heparin lyase, no EC number) has been purified from Bacteroides stercoris HJ-15, isolated from human intestine, which produces three kinds of heparinases. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, DEAF-cellulose, CM-Sephadex C-50, hydroxyapatite, and HiTrap SP chro-matographies with a final specific activity of 19.5μmol/min/mg. It showed optimal activity at pH 7.2 and 45°C and the presence of 300mM KCl greatly enhanced its activity. The purified enzyme activity was inhibited by Cu2+ Pb2+, and some agents that modify histidine and cysteine residues, and activated by reducing agents such as dithiothreitol and 2-mercaptoethanol. This purified Bacteroides heparinase is an eliminase that shows its greatest activity on bovine intestinal heparan sulfate, and to a lesser extent on porcine intestinal heparan sulfate and heparin. This enzyme does not act on acharan sulfate but de-O-sulfated acharan sulfate and N-sulfoacharan sulfate were found to be poor sub-strates. The substrate specificity of this enzyme is similar to that of Flavobacterial hepa-rinase II. However, an internal amino acid sequence of the purified Bacteroides heparinase shows significant (73%) homology to Flavobacterial heparinase III and only 43% homology to Flavobacterial heparinase II. These findings suggest that the Bacteroidal heparinase is a novel enzyme degrading GAGs.View full abstractDownload PDF (1631K) -
Hai-Hui Xue, Dong-Mei Zhao, Takafumi Sucla, Chiharu Uchida, Toshiaki O ...2000 Volume 128 Issue 2 Pages 329-336
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCapacitative Ca2+ entry is essential for refilling intracellular Ca2+ stores and is thought to be regulated primarily by inositol 1, 4, 5-trisphosphate (IP3)-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca2+ release in the absence of extracellular Ca2+ similar to that induced by thapsigargin (Tg), and Ca2+ entry occurs upon the readdition of extracellular Ca2+. The channels thus activated are also permeable to Mn2+. The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca2+ concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca2+ release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca2+ release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP3 receptor. These results suggest that in A549 cells, (i) capacitative Ca2+ entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP3, probably via Ca2+-induced Ca2+ release.View full abstractDownload PDF (1184K) -
Takanori Otomo, Toshio Yamazaki, Katsuhiko Murakami, Akira Ishihama, Y ...2000 Volume 128 Issue 2 Pages 337-344
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino-terminal domain of the α subunit (αNTD) of Escherichia coli RNA polymerase consisting of 235 amino acid residues functions in the assembly of the α, β, and β' subunits into the core-enzyme. It has a tendency to form aggregates by itself at higher concentrations. For NMR structural analysis of αNTD, the solution conditions, including the use of non-denaturing detergents, were optimized by monitoring the translational diffusion coefficients using the field gradient NMR technique. Under the optimal conditions with taurodeoxycholate and with the aid of deuteration of the sample, αNTD gave triple-resonance spectra of good quality, which allowed the assignment of a large part of the backbone resonances. Analysis of the pattern of NOEs observed between the backbone amide and α-protons demonstrated that αNTD has three α-helices and two β-sheets. Although the secondary structure elements essentially coincide with those in the crystal structure, the larger of the two β-sheets has two additional β-strands. The irregular NOE patterns observed for the three positions in the β-sheets suggest the presence of β-bulge structures. The positions of the three helices coincide with the conserved sequence regions that are responsible for the subunit assembly.View full abstractDownload PDF (1758K)
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