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Volume 118 (1995)
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Volume 118, Issue 3
Displaying 1-30 of 30 articles from this issue
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Hideyuki Hayashi1995 Volume 118 Issue 3 Pages 463-473
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPyridoxal 5'-phosphate (PLP) acts as the coenzyme in a vast number of reactions in amino acid metabolism. The study of PLP enzymes is one of the most fascinating frontiers in enzymology, and now the mechanisms of several types of PLP enzymes are being discussed at the atomic level based on crystallographic, spectroscopic, and site-directed mutagenesis studies. In this review, I summarize the important findings, including those provided by classical studies, on the reaction mechanisms of several PLP enzymes, with the intention of discussing the chemically and thermodynamically consistent principle of the catalytic action of PLP enzymes common to all the enzymes of this group, and the uniqueness of individual enzymes that endows them substrate and reaction specificity.View full abstractDownload PDF (1408K) -
Hideyuki Miyatake, Yasuo Hata, Tomomi Fujii, Kensaku Hamada, Kazuyuki ...1995 Volume 118 Issue 3 Pages 474-479
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of the unliganded alkaline protease from Pseudomonas aeruginosa IFO3080 has been determined at 2.0 Å resolution by the X-ray method. The enzyme consists of N-terminal catalytic and C-terminal β-helix domains. On structural comparison between the present unliganded enzyme and structurally-known liganded enzyme, some structural changes were observed around the active site. In the unliganded enzyme, Y216 serves as the fifth ligand for the active site zinc ion. On ligand binding, Y216 may move to form a hydrogen-bond with the carbonyl oxygen of the P1 residue of a ligand peptide. D191 in the flexible loop, Y190 to D196, over the active site cleft forms hydrogen-bonds with the backbone atoms of the P1 and P2 residues of the ligand to close the entrance of the cleft. The water molecule which is the fourth ligand for the zinc ion is replaced by the carbonyl oxygen of the P1 residue. These structural changes around the active site may reflect the substrate-binding mode during the enzymatic reaction.View full abstractDownload PDF (791K) -
Cecilia M. Devlin, Mark R. Bowles, Ross B. Gordon, Susan M. Pond1995 Volume 118 Issue 3 Pages 480-487
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProducing an effective antidote against poisoning by the herbicide paraquat (PQ) has proven to be an elusive goal. One approach that holds some promise is immunotherapy with antibody fragments. In this study we detail the production of a single chain Fv fragment (scFv) specific for paraquat by linking cloned heavy (VH) and light chain (VL) variable region genes via the peptide spacer (Gly4-Ser)3. These genes were obtained from hybridomacells secreting aPQ-specific murine monoclonal antibody. The scFv (28 kDa) was expressed at 4% of the total cell protein by the Escherichia coli vector, pPOW, but was associated with the membranes. After solubilisation and reduction, the scFv was renatured by rapid dilution. Western blotting confirmed that the refolded scFv had similar structural properties to the parental mAb. The isoelectric point of the scFv (7.0) is equal to the value calculated from the deduced amino acid sequence. Surface plasmon resonance was used to demonstrate specific PQ binding by the refolded scFv (Ka=1.24×106M-1) which is similar to that determined for the parent Fab fragment (4.6×106M-1).View full abstractDownload PDF (1927K) -
Hidenori Shinkawa, Nobuyuki Fujita, Tetsuo Shiina, Kan Tanaka, Hideo T ...1995 Volume 118 Issue 3 Pages 488-493
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRNA polymerase was purified from vegetative-phase mycelia of Streptomyces griseus by a series of ion-exchange chromatographies. By western blot analysis using antiserum against S. coelicolor HrdB, which is a principal sigma factor (σhrdB), the purified holoenzyme was found to contain σB (=σhrdB) of S. griseus. Significant amounts of HrdB protein were, however, eluted from the DEAE column at lower concentrations of KCl than that required for elution of the holoenzyme containing σB, suggesting that σB is dissociated from the core enzyme, or an excess amount of σB exists in S. griseus cells. The holoenzyme containing σB (EσB) transcribed in vitro the dagA promoter of S. coelicolor, and the hrdB and hsp7O promoters of S. griseus, suggesting that it is involved in transcription of the essential genes. EσB may be a major form of RNA polymerase holoenzyme in the growing phase of S. griseus.View full abstractDownload PDF (4566K) -
Hidenori Shinkawa, Yuji Hatada, Masato Okada, Haruyasu Kinashi, Osamu ...1995 Volume 118 Issue 3 Pages 494-499
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hrdB homologue was isolated from a streptomycin-producing Streptomyces griseus 2247 strain, which is independent of A-factor. The nucleotide sequence of the cloned DNA fragment revealed the presence of an open reading frame (ORF) of 1, 542 bp, which predicted a primary product of 514 amino acids and Mr 56, 100. The N-terminal sequence of the purified HrdB protein of S. griseus was identical to the amino acid sequence deduced from the nucleotide sequence. The deduced amino acid sequence contains an “rpoD box” conserved in the principal sigma factors of eubacteria, and shows high similarity to the hrdB products of S. coelicolor A3 (2) (89.9%) and S. aureofaciens (88.1%). The cloned gene encodes a principal sigma factor of S. griseus. The promoter region was identified by using a promoter-probe vector and by means of primer extension experiments. The transcription start point is located 158-bp upstream of the initiation codon.View full abstractDownload PDF (1608K) -
Takashi Agui, Xiaonan Xin, Yiqiang Cai, Giljin Shim, Youji Muramatsu, ...1995 Volume 118 Issue 3 Pages 500-507
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe regulation of the gene expression of the atrial natriuretic peptide receptor (ANPR) subtypes, ANPR-A, ANPR-B, and ANPR-C, was investigated in a murine thymic stromal cell line, MRL 104.8a. When MRL 104.8a cells were cultured with transforming growth factor (TGF)-β1, [125I] ANP binding sites increased with increasing dose of TGF-β1. These binding sites were identified as ANPR-C by a displacement experiment with ANPR-C-specific ligand, C-ANF, and by the affinity cross-linking of the [125I] ANP binding sites with a chemical cross-linker to determine the molecular weight of the ANPR. This augmentation of the ANPR-C expression was elucidated to occur at the transcriptional level by Northern blot experiment, comparison of the relative amounts of mRNA by reverse transcription (RT)-PCR, and in vitro nuclear transcription assay. Conversely, the expression of the ANP biological receptors, ANPR-A and ANPR-B, was shown to be down-regulated by TGF-β1. These data suggest that TGF-β1, regulates the gene expression of ANPRs in the thymic stromal cells and that ANP and TGF-β1, might affect the thymic stromal cell functions.View full abstractDownload PDF (4438K) -
Takao Ohyashiki, Kanae Kamata, Miyako Takeuchi, Katsuhiko Matsui1995 Volume 118 Issue 3 Pages 508-514
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExposure of rat liver microsomes to ascorbic acid/Fe2+ caused decreases in the membranebound glucose-6-phosphatase (G-6-Pase) activity and the protein thiols after a short lag period (4min). Under the same conditions, the production of thiobarbituric acid-reactive substances and fluorescent products was also initiated from 4 min after the start of the treatment, although conjugated diene was formed immediately on incubation of the microsomes with ascorbic acid/Fe2+. After centrifugation of the treated microsomes, the fluorescent products and the enzyme activity remained in the membrane fraction. The results of kinetic studies of the enzyme activity indicated that ascorbic acid/Fe2+-induced inhibition of the enzyme activity is mainly due to an increased Km value for the substrate. A decreased activity of the microsomal G-6-Pase was also observed when the microsomes were incubated with aldehydes such as malondialdehyde, n-heptaldehyde, acetaldehyde, and trans-2-nonenal. However, loss of protein thiols was detected only upon treatment of the microsomes with trans-2-nonenal, Glucose-6-phosphate (G-6-P) effectively prevented ascorbic acid/Fe2+- or trans-2-nonenal-induced inhibition of the enzyme activity, but the substrate failed to protect the protein thiols in both systems. The results of fluorescence anisotropy measurements of diphenylhexatriene-labeled microsomes suggested that changes in the lipid dynamics are not directly related to peroxidation-mediated inhibition of the enzyme activity. Based on these results, a possible reason for the inhibition of the microsomal G-6-Pase activity associated with ascorbic acid/Fe2+ treatment is discussed.View full abstractDownload PDF (913K) -
Yasushi Shigeri, Shigeyuki Nakajima, Masafumi Fujimoto1995 Volume 118 Issue 3 Pages 515-520
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn porcine aortic smooth muscle cells, neuropeptide Y (NPY) stimulates mobilization of Ca2+ from intracellular store sites via Y1 receptors. However, it has been debated whether or not Ca2+ mobilization by Y1 receptors depends on the generation of inositol 1, 4, 5-trisphosphate [Ins(1, 4, 5)P3] following activation of phospholipase C. To examine this question, we studied the effect of U73122, an inhibitor of phospholipase C-mediated inositol phosphate accumulation, on the NPY-induced rise in cytosolic free Ca2+ concentration ( [Ca2+]i) in comparison with that on angiotensin II (All)-induced [Ca2+]i, increase, which is dependent on Ins(1, 4, 5)P3 generation. Digital-imaging microscopy study using the Ca2+ sensitive dye fura-2 revealed that application of All induced a rapid but transient [Ca2+]i increase in a single cell, arising from intracellular calcium stores. Application of NPY to the same cell induced a [Ca2+]i rise with a pattern similar to that induced by All. All increased the formation of Ins(1, 4, 5)P3 by about 3.0-fold, while the NPY-induced formation was very small. U73122 completely inhibited not only Ins(1, 4, 5)P3 synthesis, but also Ca2+ mobilization induced by either agonist. The effect of U73122 on the NPY-induced [Ca2+]i increase was about 10-fold more potent than that on the All-induced one. U73343, an inactive analog of U73122, had no influence on any of the All- and NPY-mediated effects. Herbimycin A completely inhibited the platelet-derived growth factor-induced [Ca2+]i increase, but had no effect on the NPY-induced [Ca2+]i increase, indicating that phospholipase C-γ is not involved in the NPY effect. These results suggest that NPY-induced Ca2+ mobilization from intracellular stores in porcine smooth muscle cells is secondary to the very small generation of Ins(1, 4, 5)P3 following stimulation of phospholipase C-β, which may account for the hypersensitivity of the NPY effect to U73122.View full abstractDownload PDF (1616K) -
Yoshiro Saito, Hideharu Ikebuchi, Takeshi Yamazaki, Jun-ichi Sawada1995 Volume 118 Issue 3 Pages 521-525
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSoluble forms of growth hormone receptors, growth hormone-binding proteins (GH-BPs), with molecular weights of 60 and 55 kDa were found to be constitutively released from human IM-9 cells. The release of the GH-BPs was not inhibited by inhibitors of protein synthesis (cycloheximide) and transport (brefeldin A). Down-regulation by human growth hormone or trypsin pretreatment of surface growth hormone receptors abolished the GH-BP release, suggesting that cell-surface human growth hormone receptors are involved in the GH-BP release. Several inhibitors of serine-, thiol-, and acid-proteases did not affect the GH-BP release. EDTA efficiently blocked the GH-BP release. This inhibition by EDTA was restored by addition of Mg2+ and Co2+. These results suggest that human GH-BPs are constitutively released by proteolytic cleavage of cell-surface growth hormone receptors by a metalloprotease.View full abstractDownload PDF (4638K) -
Tadashi Ii, Yoko Ohashi, Shigeki Nunomura, Tomoya Ogawa, Yoshitaka Nag ...1995 Volume 118 Issue 3 Pages 526-533
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructural characterization of mono-, di-, and trisulfated trisaccharides by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of sulfated trisaccharides allowed identification of the sulfated sugar and provided information on the saccharide chain sequence. The ESI CID-MS/MS of multiple-charged ions provided even more structural information, and some of the useful daughter ions appeared in the higher mass region than the precursor owing to a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated analogs.View full abstractDownload PDF (741K) -
Toshie Shinagawa, Hiroyuki Nunoi, Shigeaki Nonoyama, Shiro Kanegasaki1995 Volume 118 Issue 3 Pages 534-540
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe antigenic protein CD4O on the surface of B lymphocytes plays an important role in their proliferation, immunoglobulin class switching, and rescue from apoptosis in the germinal center through interaction with T lymphocytes expressing CD40 ligand. The protein is also found on the cell surface of other antigen-presenting cells such as monocytes, dendritic cells, and thymic epithelium cells, but its presence in other myeloid cells has not been reported. We show here that CD40 protein is induced in promyelocytic HL60 cells, when cultured with retinoic acid, a vitamin that converts them to granulocyte-like cells. The cultured cells also expressed CD15, a marker for granulocytes, and cytochrome b558, an essential component of the superoxide-generating system in phagocytes, on their surface. No detectable amount of mRNA for CD40 was found in naive HL60 cells, whereas a large amount of the message was induced in the cells cultured with the vitamin. Although CD40 expression was enhanced when the cells were further cultured with GM-CSF or IFN-γ, expression of CD14, a marker for monocytes, was also enhanced. HL60 cells, therefore, express CD40 protein during differentiation not only toward monocytes but also toward granulocytes, at least transiently.View full abstractDownload PDF (2126K) -
Hideki Masutani, Hiroshi Kimura1995 Volume 118 Issue 3 Pages 541-545
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-(1, 2)-Fucosyltransferase (GDP-L-fucose: β-D-galactoside 2-α-L-fucosyltransferase) from human gastric mucosa was purified to homogeneity by column chromatographies on Ultrogel AcA34, phenyl-Sepharose, hydroxylapatite, SP-Sephadex, and GDP-hexanolamine Sepharose. The molecular weight of the purified enzyme was estimated to be 65, 000 by SDS-PAGE. The Km value of this enzyme for a type 1 sugar acceptor was a little smaller than that for a type 2 one, indicating this enzyme is a secretor-type α-(1, 2)-fucosyltransferase. However, the difference between the Km value for a type 1 precursor and that for a type 2 one was very small, suggesting that this enzyme can use both types of precursors as sugar acceptors approximately equally, unlike the purified α-(1, 2)-fucosyltransferase from human serum as the secretor-type reported previously. The characteristics of the purified enzyme were compared with those of H-type α-(1, 2)-fucosyltransferase from human plasma. The activities of both enzymes were inhibited by salt and N-ethylmaleimide, but they showed a significant difference in their divalent cation requirements.View full abstractDownload PDF (1779K) -
Yuji Ito, Miyuki Nakamura, Takuji Hotani, Taiji Imoto1995 Volume 118 Issue 3 Pages 546-551
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLysozyme was purified from the homogenate of the whole body of house fly (Musca domestica) larvae by standard chromatographic techniques. The purified lysozyme was sequenced and its enzymatic properties were examined. This lysozyme was a chicken-type lysozyme composed of 122 amino acids, showing about 75% identity with fruit fly lysozymes and 38% with human lysozyme. This enzyme was inactive towards Micrococcus luteus under the physiological conditions of pH 7.0 and ionic strength 0.1, but was as active towards glycol chitin as was hen lysozyme. The pH-dependent profile of lytic activity towards M. luteus showed that house fly lysozyme has an acidic pH optimum and shows no enzymatic activity above pH 7. These features are analogous with those of ruminant stomach lysozymes which have evolved for the digestive function, suggesting that this lysozyme does not function as a self-defense protein, like hen and human lysozyme, but as a digestive enzyme, probably in the gut of the insect body. Although a similar functional conversion to digestive enzyme was reported in fruit fly, phylogenetic tree analysis indicates that the evolutionary change of lysozyme to a digestive enzyme occurred similarly in fruit fly and house fly, but the events are not related and occurred independently in each strain. This observation is in contrast with the case of ruminant stomach lysozymes, which were recruited before the divergence of each species of ruminants.View full abstractDownload PDF (623K) -
Atsushi Sakurai, Hideki Horibe, Nobuhiro Kuboyama, Yuji Hashimoto, Yas ...1995 Volume 118 Issue 3 Pages 552-554
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to determine the absolute configuration of urothion (1), the CD spectra of tri-4-chlorobenzoyl derivatives of 2-amino-6-[(3R)-3, 4-dihydroxybutyl]pteridin-4 (3H)-one and its (3S) compound were compared with that of the tri-4-chlorobenzoyl derivative obtained from the natural product. R-Configuration was concluded for the secondary hydroxyl group on the side chain of 1, which is the same configuration as that of molybdopterin (2). This supports the view that 1 might be a urinary metabolite of 2.View full abstractDownload PDF (268K) -
Hideki Adachi, Masafumi Tsujimoto1995 Volume 118 Issue 3 Pages 555-561
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene encoding dipeptidase was cloned from Acinetobacter calcoaceticus ATCC 23055. Determination of the nucleotide sequence revealed that the gene had an open reading flame of 1, 050 by coding a protein of 350 amino acids. The deduced amino acid sequence showed 48.8% similarity to human renal dipeptidase and conserved two amino acid residues identified in human and pig renal dipeptidases as essential ones for the catalytic activity. Purified recombinant enzyme expressed in Escherichia coli did not hydrolyze the unsaturated dipeptide, glycyldehydrophenylalanine. On the other hand, it preferentially hydrolyzed dipeptides having a D-amino acid, when compared with those having an L-amino acid at the C-terminal. Furthermore, it could not hydrolyze tripeptides. These results indicate that the dipeptidase produced by A. calcoaceticus ATCC 23055 has a unique substrate specificity and preferentially hydrolyzes dipeptides having a D-amino acid at the C-terminal.View full abstractDownload PDF (4351K) -
Naohiro Yoshigi, Yukio Okada, Hideo Maeba, Hirohisa Sahara, Teruo Tama ...1995 Volume 118 Issue 3 Pages 562-567
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo increase the thermostability of β-amylase, seven kinds of single-mutant plasmids were constructed with an expression vector of barley β-amylase by mutagenesis. The remaining activity versus temperature curves were used to determine the temperatures (T50) at which 50% of the initial activity was lost during a 30-min heating period. These mutations increased the T50 values by amounts ranging from 0.8 to 3.2°C. To express the sevenfoldmutant β-amylase in Escherichia coli, plasmid pB927 was constructed. E. coli harboring plasmid pB927 produced sevenfold-mutant β-amylase. The T50 value of purified sevenfoldmutant β-amylase (69.0°C) was higher than that of not only the original recombinant β-amylase (57.4°C) by 11.6°C but also soybean β-amylase (63.2°C) by 5.8°C. The intragenic amino acid replacements were found to have simple additive effects on the thermostability of β-amylase. The sevenfold-mutant β-amylase was found to be stable at pHs up to 12.5, while the original recombinant β-amylase was unstable at pHs above 9.5. The data obtained from kinetics studies suggested that the sevenfold-mutant β-amylase acquired enhanced thermostability, but its function as a β-amylase remained unchanged.View full abstractDownload PDF (2745K) -
Janet A. Meurer, Joan M. Naylor, Carolyn A. Baker, Darrell R. Thomsen, ...1995 Volume 118 Issue 3 Pages 568-574
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOligonucleotide primers derived from the eDNA encoding a full-length bovine UDPGalNAc: polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) [Homa, F. L., Hollander, T., Lehman, D. J., Thomsen, D. R., and Elhammer, Å. P. (1993) J. Biol. Chem 268, 12609-12616], were used for PCR to isolate sequences encoding a homologous enzyme from human salivary gland cDNA. Comparison of the human and bovine nucleotide sequences reveals 94.8% sequence identity in their coding regions and 87% identity in their 3-untranslated regions. The translation of the human GalNAc-transferase coding region predicts an amino acid sequence which is nearly identical (99.6%) to that of the bovine counterpart; there are five conservative and one non-conservative amino acid substitutions between the two enzymes. Expression of the bovine and human cDNAs in the insect cell line, Sf9, resulted in the synthesis of proteins which appeared identical on SDS-PAGE and which had similar enzymatic properties. Screening of a somatic cell human/rodent hybrid panel with a probe derived from the human GalNAc-transferase cDNA sequence indicated that the human GalNAc-transferase gene is localized to chromosome 18.View full abstractDownload PDF (3204K) -
Audrey H. Hobson, Catherine M. Buckley, Steen T. Jørgensen, B&o ...1995 Volume 118 Issue 3 Pages 575-581
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lipA gene of Pseudomonas cepacia DSM3959 requires a downstream gene, limA, in order to express lipase activity. The product of the limA gene, LimA, is a molecular chaperone required during the folding of lipase in order for the lipase to adopt an active conformation. The lipase and LimA proteins have been shown to form a complex precipitable with either an anti-lipase or anti-LimA antibody. LimA has been shown to form a 1:1 complex with prelipase and lipase isolated from “natural” P. cepacia system. The mature lipase (lacking its signal peptide) has been expressed in the presence and absence of limA in Escherichia coli. LimA can activate mature lipase during a urea denaturation-renaturation experiment, indicating that the signal peptide is not required for the lipase to be activated by LimA. The effects of various reagents on the renaturation of lipase from 8M urea have been examined. We propose a mechanism for the function of the LimA chaperone during the production of active extracellular lipase.View full abstractDownload PDF (2445K) -
Hiroshi Wachi, Yoshiyuki Seyama, Shingo Tajima1995 Volume 118 Issue 3 Pages 582-586
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of heparin on elastin expression in the proliferating and quiescent phases of growth of smooth muscle cells was studied. Heparin stimulated elastin synthesis and its mRNA level 2-3-fold in the proliferating cells while it inhibited the cell proliferation. The inhibition of cell proliferation and the stimulation of elastin expression by heparin in the proliferating cells were mimicked by a potent protein kinase C antagonist, H-7, but not by H-89, W-7, and HA1004, suggesting that the effect of heparin is mediated by the inhibition of protein kinase C. In contrast, heparin inhibited elastin synthesis and its mRNA level slightly but exhibited no effect on cell proliferation in the growth-arrested cells. This result indicates that heparin reciprocally affects elastin expression depending on the growth state of smooth muscle cells. Heparin thus exerts a complex influence on elastin expression in smooth muscle cells.View full abstractDownload PDF (1602K) -
Tetsuo Kobayashi, Sadaharu Higuchi, Kinuko Kimura, Toshiaki Kudo, Koki ...1995 Volume 118 Issue 3 Pages 587-592
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThermococcus profundus, a hyperthermophilic archaeon, did not exhibit detectable glutamine synthetase activity, although the organism possessed an extraordinarily high level of glutamate dehydrogenase (GDH), the content of which reached over 10% of total soluble proteins. This GDH was purified to homogeneity. The enzyme had a molecular weight of 263, 000 and was composed of six homogeneous subunits of molecular weight 43, 000. The enzyme was extremely thermostable with a half life of 1 h at 90°C. Circular dichroism (CD) spectra of the enzyme revealed gradual unfolding of α-helices upon exposure to increasing temperature. The enzyme reaction was strongly biased toward glutamate formation. T. profundus excreted L-alanine into the medium, and the concentration reached 1.5mM. High activity of alanine aminotransferase (AAT) was present in the cells, while no alanine dehydrogenase activity was detected. The alanine formation may be initiated by ammonia uptake by GDH followed by aminotransfer from glutamate to pyruvate by AAT.View full abstractDownload PDF (2052K) -
Masanori Terajima, Yasuhisa Nemoto, Masuo Obinata1995 Volume 118 Issue 3 Pages 593-600
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCis-acting elements of the gene for mouse glycophorin, an erythroid-specific membrane glycoprotein, were determined by transient and stable transfection assays using murine erythroleukemia (MEL) cells. Cis-acting elements proximal to the transcription start site of the gene can be separated into the basal promoter (-1 to -91 bp) and the distal element (-133 to -92). The basal promoter contained GGTGG and GATA motifs and the distal element contained GATA-1 and NF-E2 motifs. Deletion analysis of the distal GATA site and its neighboring sequence and DNase-I footprinting/EMSA (electrophoretic mobility shift assay) analysis indicated that induced nuclear factor binding to GATA-1 and its neighboring sequence may be required for expression during MEL cell differentiation induced by dimethyl sulfoxide treatment. The NF-E2 site was also shown to be essential for the promoter activity. An approximately 400 by far upstream region (-1325 to -948 bp) containing the binding motifs for GGGTGG, GATA-1 and NF-E2 showed no enhancing activity when this region was examined by transient transfection assay, but it did show enhancement of the differentiation-specific promoter activity in the stable transfection assay. The far upstream region of mouse glycophorin gene may have a function similar to that of the locus control region (LCR) of human β-globin gene cluster.View full abstractDownload PDF (3260K) -
Kazuo Hashido, Yuji Arai, Susumu Kajihara, Kei-ichiro Joh, Hitomi Yats ...1995 Volume 118 Issue 3 Pages 601-606
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to understand the molecular mechanisms of the temporal and spatial differences of gene expression in higher organisms, rat aldolase A gene carrying two distinct promoters was introduced into fertilized eggs and the resulting transgenic mice were analyzed. The transgene expression is tissue-specific and is developmentally regulated. In addition, the expression is regulated in a copy-dependent manner irrespective of where the transgene is integrated, suggesting that a mechanism excluding the effect of the integration site exists within the transgene itself. To explore the conformational change of this gene in the genome, the DNase I hypersensitive sites of the gene were examined. Three sites (DHS-1, 2, and 3) were identified upstream and downstream of the gene and these sites were retained in the transgene as well as in the gene observed endogenously.View full abstractDownload PDF (4825K) -
Masaharu Takeda, Takahito Okushiba, Takaaki Satoh, Shinren Kuniyoshi, ...1995 Volume 118 Issue 3 Pages 607-613
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChromosome fragmentation, ATP1 disruption, and Southern blot analyses of total DNAs and prime clones of chromosome II showed that three identical ATP1s are present, directing from the telomere to the centromere on the 35-55 kb far from the left telomere sequence of chromosome II. That is, the coding and 5'-, 3'-non-coding regions of ATP1 are repeated 3 times at approximately 7kb intervals. These three ATP1s are expressed, and one and two ATP1s-disrupted strains respectively, showed ca. 70 and 40% decreases in their ATPase activities and α subunit contents, compared to those of the wild-type, DC-5 or W303-1A strain, but could grow on glycerol.View full abstractDownload PDF (5778K) -
Yasuzo Nishina, Kyosuke Sato, Retsu Miura, Kiyoshi Shiga1995 Volume 118 Issue 3 Pages 614-620
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structure of the ligand in a charge-transfer complex of oxidized D-amino acid oxidase (DAO) formed upon aerobic addition of chloropyruvate and ammonium sulfate was determined by resonance Raman spectroscopy. The ligand in the complex is in the enamine form, 2-amino-3-chloroacrylate, not in the imine form, 2-imino-3-chloropropionate. This conclusion is consistent with our hypothesis that the ligands in the charge-transfer complexes of oxidized DAO and reduced DAO are generally in the enamine and imine forms, respectively. The calculation of HOMO and LUMO of some enamine and imine forms by the extended Htickel molecular orbital method indicated that the enamine form is a better electron donor than the imine form and the imine form is a better electron acceptor than the enamine form. These results, as well as the information about the structure of enzymebound ligand, support the following ideas. (i) In the charge-transfer complex of oxidized DAO, the oxidized flavin is an electron acceptor and the ligand is an electron donor. (ii) In the charge-transfer complex of reduced DAO, the reduced flavin is an electron donor and the ligand is an electron acceptor. Resonance Raman studies on the charge-transfer complexes of oxidized DAO and those of reduced DAO, and calculated results of HOMO and LUMO for some enamine and imine forms, corroborated the structure of the stacking form between the flavin ring and the ligand in the active site of DAO.View full abstractDownload PDF (828K) -
Takashi Sakai, Takahisa Furukawa, Hiroko Iwanari, Chio Oka, Toru Nakan ...1995 Volume 118 Issue 3 Pages 621-628
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRBP-Jx is a novel type of transcriptional regulatory protein that does not contain any known DNA-binding motif. We raised anti-RBP-Jx monoclonal antibodies (K0043 and T6709) to investigate the roles of RBP-Jx in cell differentiation. These antibodies stained nuclei of undifferentiated embryonic stem cells and F9 cells but not those of the other differentiated cell lines tested so far although the RBP-Jx protein is expressed at similar levels. Interestingly, differentiated F9 cells lost the immunostaining reaction with the antiRBP-Jx monoclonal antibodies. Biochemical subcellular fractionation study showed that the majority of RBP-Jx was localized in nuclei of F9 cells and that there are at least two forms of the RBP-Jx protein in the nuclei of undifferentiated F9 cells, a free form and a chromatin-bound form. Upon induction of F9 cell differentiation, free nuclear RBP-Jx disappeared concomitantly with the loss of immunostaining, suggesting that the anti-RBP-Jx antibodies cannot recognize chromatin-bound RBP-Jx. Since there is no evidence to indicate covalent modification of RBP-Jx, we assume that chromatin-bound RBP-Jx interacts with a large number of proteins which block the exposure of RBP-Jx epitopes to the monoclonal antibodies.View full abstractDownload PDF (5620K) -
Hidenori Ito, Kaori Hasegawa, Yutaka Inaguma, Osamu Kozawa, Tomiko Asa ...1995 Volume 118 Issue 3 Pages 629-634
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe expression of αB crystallin, hsp27, and hsp70 in C6 cells increased when the cells were exposed to arsenite (50μM for 1h) or heat (42°C for 30min), as detected by specific immunoassays, Western blot analysis, and Northern blot analysis. When cells were exposed to arsenite in the presence of 0.1μM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or 0.2μM okadaic acid, an inhibitor of phosphoserine/phosphothreonine protein phosphatases, expression of αB crystallin was markedly enhanced. The induction of hsp27 and hsp70 expression was also stimulated to a considerable extent in the same cells. The stimulatory effect of PMA was further enhanced in the presence of okadaic acid, but it was strongly inhibited in the presence of 0.5μM staurosporine, an inhibitor of protein kinase C. PMA and okadaic acid also stimulated the response to heat stress of the expression of αB crystallin, but they barely stimulated the response to heat stress of hsp27. The extent of stimulation of the arsenite-induced responses by PMA and okadaic acid was greater when the concentration of arsenite (i.e. the magnitude of the stress) was relatively low (25-50μM). The arsenite-induced release of arachidonic acid from cells was also stimulated in the presence of PMA and/or okadaic acid, and the stimulatory effects of PMA and okadaic acid on the arsenite-induced accumulation of αB crystallin and hsp27 were strongly suppressed by quinacrine, an inhibitor of phospholipase A2. These results suggest that the stimulatory effects of PMA and okadaic acid on the stress responses are caused, in part, by the increased metabolic activity of the arachidonic acid cascade, as a consequence of the activation of phospholipase A2.View full abstractDownload PDF (716K) -
Kazunori Shibuya, Hiroyuki Kaji, Yukihito Ohyama, Shin-ichi Tate, Masa ...1995 Volume 118 Issue 3 Pages 635-642
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe expression system for human recombinant cystatin A has already been established to be a fusion protein with porcine adenylate kinase in Escherichia coli [Kaji et al. (1990) Biol. Chem. Hoppe-Seyler 371, Suppl., 145-150]. After cyanogen bromide cleavage of the fused protein expressed in E. coli, the cystatin portion could be readily isolated. The inhibitory activity of the obtained variant (Cyst A2-98) was found to be almost identical with that of the wild type, and thereafter a mutation was introduced into this variant (Cyst A2-98), called the standard variant. To elucidate the role of the Gly-4 residue, which is completely conserved in all cystatin species, this residue was substituted with 17 other amino acids by means of cassette mutagenesis. Thus 17 variants (Cyst A2-98 [G4X]) obtained were examined as to their inhibitory activity towards papain. As the side chain of the substituted amino acid residue became more bulky, the inhibitory activity of the variant markedly decreased. Variants whose side chains were bulkier than a Val residue showed almost no inhibitory activity towards papain. Consequently, it was deduced that the large side chain of a substituted amino acid may cause steric hindrance, which may be responsible for the decrease in inhibitory activity. Thus, we could conclude that the 4th (Gly) residue on cystatin A must be small, because amino acids which existed on the N-terminal side of this residue could interact with a papain molecule.View full abstractDownload PDF (1892K) -
Yasunobu Matsuda, Kunio Matsumoto, Takafumi Ichida, Toshikazu Nakamura1995 Volume 118 Issue 3 Pages 643-649
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHepatic fibrosis/cirrhosis is a common hepatic disease characterized by the hyper-accumulation of connective tissue components, and hepatic necrosis. Chronic alcohol ingestion, viral infection, and metabolic disorders are contributing factors and there has been no effective treatment. Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature hepatocytes, is a long-sought hepatotrophic factor for liver regeneration. Administration of human recombinant HGF into rats with hepatic fibrosis/cirrhosis caused by dimethylnitrosamine (DMN) elicited mitogenic action for hepatocytes, stimulated hepatic collagenase activity, and prevented the onset and progression of hepatic fibrosis/cirrhosis. Accumulation of fibrous tissue components in the liver due to DMN-treatment were markedly decreased in HGF-injected rats. Moreover, HGF completely abrogated death caused by severe hepatic cirrhosis and dysfunction. We postulate that HGF may prove to be an effective treatment for human liver fibrosis/cirrhosis and for chronic hepatic failure.View full abstractDownload PDF (1891K) -
Shin-ichi Inoue, Shin-ichi Hoshino, Iwao Kukimoto, Michio Ui, Toshiaki ...1995 Volume 118 Issue 3 Pages 650-657
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe expressed the Gly203→Thr (G203T) mutant of Gi2α, which was expected to show a dominant-negative phenotype in Gi2-mediated signal transduction, in baculovirus-infected Sf9 cells and purified the mutant α subunit for its characterization. The rate of dissociation of GDP from G203T Gi2α was 3- to 4-fold faster than that from wild-type Gi2α, but their kcat values for GTP hydrolysis were almost the same. The affinities of the two Gi2α proteins for the βγ subunits of G proteins to form αβγ trimers, which served as substrates for pertussis toxin-catalyzed ADP-ribosylation, were the same. In marked contrast, G203T Gi2α was unable to form a tight complex with a non-hydrolyzable analog (GTP[γS]) of GTP; bound GTP[γS] was readily released from the mutant Gi2α even in the presence of a high concentration of Mg2+. Its susceptibility to tryptic digestion also revealed that GTP [γS]-bound G203T Gi2α formed a conformation apparently different from that of the GTP[γS]-bound form of wild-type Gi2α. Both the G203T and wild-type Gi2α proteins were capable of coupling with membrane-bound α2-adrenergic receptors, resulting in the formation of receptor-G protein complexes with high affinity for agonists. However, GTP [γS] -dependent uncoupling from the high-affinity receptors was markedly attenuated in the case of G203T Gi2α. Thus, G203T-mutated Gi2α had a unique property in terms of coupling to membrane receptors, in addition to the previously expected defect in the active conformation of the GTP-bound form of Gi2α.View full abstractDownload PDF (2207K) -
Yukiko Yoshida, Naoya Kojima, Shuichi Tsuji1995 Volume 118 Issue 3 Pages 658-664
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA encoding a new α2, 8-sialyltransferase (ST8Sia IV), which exhibits activity toward the α2, 3-linked sialic acids of N-linked oligosaccharides, was cloned from a mouse lung cDNA library by means of the PCR-based approach. The predicted amino acid sequence of ST8Sia IV showed 15.2, 56.0, and 26.2% identity with those of so far cloned mouse α2, 8-sialyltransferases, i.e., GD3 synthase (ST8Sia I), STX (ST8Sia II), and Siaα2, 3Galβ1, -4G1cNAc α2, 8-sialyltransferase (ST8Sia III). ST8Sia IV exhibits high amino acid sequence identity (99.2%) with recently cloned hamster polysialyltransferase-1 gene, which is necessary to polysialic acid expression, but no enzymatic activity of the gene product was reported [Eckhardt, M. et al. (1995) Nature 373, 715-718]. The ST8Sia IV gene was strongly expressed in lung, heart, and spleen, but only weak expression of the gene was observed in brain, without remarkable developmental regulation. The activity of mouse ST8Sia IV was specific toward sialylated glycoproteins. The linkage-specific sialidase treatment of glycoproteins as well as N-linked oligosaccharides from the glycoproteins revealed that ST8Sia IV exhibits an α2, 8-sialyltransferase activity toward α2, 3-linked sialic acids of N-linked oligosaccharides. In addition, ST8Sia IV can synthesize polysialic acid chain in vitro without any initiator sialyltransferase.View full abstractDownload PDF (3136K)
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