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Volume 127 (2000)
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Volume 127, Issue 2
Displaying 1-22 of 22 articles from this issue
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Takashi Muramatsu2000 Volume 127 Issue 2 Pages 171-176
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSKnockout mice lacking glycosyltransferases or sulfotransferases provide unequivocal evidence that the carbohydrate moieties of glycoproteins, glycolipids, and proteoglycans play essential roles in various biological phenomena such as development, the immune response, and tissue functions. Examples of abnormalities of null mutants include arrest of embryogenesis due to deletion of N-acetylglucosaminyltransferase I or glucosylceramide synthase, failure of kidney formation in heparan sulfate 2-O-sulfotransferase deficiency, suppressed antibody production in α-2, 6-sialyltransferase deficiency, male sterility in GM2/GD2 synthase deficiency, and abnormalities in the function and stability of myelin in galactosylceramide deficiency.View full abstractDownload PDF (644K) -
Yasuji Yamashita, Kazuhiko Takeshige, Atsuto Inoue, Shigehisa Hirose, ...2000 Volume 127 Issue 2 Pages 177-179
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe roles of natriuretic peptides in cardiovascular homeostasis have been well characterized. A recent study revealed that mice lacking natriuretic peptide receptor-C (NPRC) exhibit skeletal-overgrowth. We, therefore, performed in situ hybridization with riboprobes to determine the localization of mRNAs for receptors for natriuretic peptides in the growth plate of the fetal mouse tibia The amount of mRNA for NPR-A was below the detectable level in the growth plate. The mRNA for NPR-B was detected predominantly in proliferating chondrocytes. By contrast, high levels of mRNA for NPR-C were found in hypertrophic chondrocyies. In other regions of the growth plate, the levels of mRNA for NPR-C were very low. The patterns of expression of mRNAs for NPR-B and NPR-C, namely, subtype switching during differentiation from proliferating chondrocytes to hypertrophic chondrocytes, suggest that these receptors might be involved in the growth and differentiation of the growth plate during fetal development in the mouse.View full abstractDownload PDF (1291K) -
Prasanta Chakraborty, Debjani Ghosh, Mukul K. Basu2000 Volume 127 Issue 2 Pages 185-190
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPretreatment of macrophages with PMA, an agonist of PKC, showed diverse effects on degradation and survival of two virulent strains of Leishmania donovani promastigotes. Treatment of macrophages with PMA for 45min at 37°C generated significant amounts of superoxide anions and reduced the parasite burden of macrophages by up to 48 and 43% when AG 83 and GE-1 strains were used for infection. Staurosporine, an inhibitor of PKC, inhibited PMA-dependent killing of the parasites, while tyrphostin AG 126, an inhibitor of protein tyrosine kinase, showed very little effect. Depletion of PKC by prolonged incubation with PMA drastically reduced the superoxide anion generation and increased the uptake and multiplication of the parasites. Finally, to understand the mechanism of higher uptake of the parasites by PKC-depleted macrophages, membrane microviscosity was measured by fluorescence depolarization. Membrane microviscosity was found to be approximately 40% lower in PKC-depleted macrophages than in normal macrophages, indicating the role of membrane fluidity in the infection process. Together, these data suggest PKC activation, superoxide generation, and membrane fluidity are essential factors in the efficient regulation of leishmanial infection.View full abstractDownload PDF (685K) -
Shinsaku Maruta, Junya Saitoh, Tsuyoshi Asakura2000 Volume 127 Issue 2 Pages 199-204
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecent crystallographic studies have shown that smooth muscle myosin has three highly conserved unique loops, loop B (320-327), loop M (687-699), and loop N (125-134), similar to other myosins, skeletal muscle and dictyostelium myosins. We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino acids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, in order to clarify the role of these smooth muscle myosin loops in energy transduction, we specifically labeled the loops with a fluorescent photoreactive ADP analogue, 3'-O-(N-methylanthraniloyl)-8-azido-ADP (Mant-8-N3ADP), and then measured the fluorescent polarization. When Mant-8-N3-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant-S-N3 ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8-N3-ADP trapped by beryllium fluoride was covalently incorporated into both loop M (687-699) and loop N (125-134) at an almost equimolar ratio. Actin binding to smooth muscle myosin S1 (SMO-S1) labeled at only loop N (125-134) increased the polarization due to the viscosity of actin. In contrast, S1 labeled at both loops N and M showed a much smaller increase in polarization. Our results indicate that the probe at loop M (687-699) of smooth muscle myosin moved to a less hindered region, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677-689) in skeletal muscle myosin, as previously demonstrated in our laboratory.View full abstractDownload PDF (2976K) -
Bae Dong Jung, Kazuhiro Kimura, Hiroshi Kitamura, Kennedy Makondo, Kei ...2000 Volume 127 Issue 2 Pages 205-209
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNon-invasive immobilization stress causes an increase in the plasma interleukin (IL)-6 level accompanied by increased IL-6 mRNA expression and IL-6 immunoactivity in the liver [Biochem. Biophys. Res. Commun. (1997) 238, 707-711]. In the present study, using rat primary cultured hepatocytes and non-parenchymal liver cells, the effect of norepinephrine (NE) on IL-6 mRNA expression was determined. IL-6 mRNA expression in hepatocytes, but not in non-parenchymal liver cells, increased when the cells were treated with NE. The stimulatory effect of NE was inhibited by the combined use of α-and β-adrenergic antagonists. IL-6 mRNA expression in hepatocytes also increased on incubation with the culture medium of non-parenchymal liver cells treated with NE. The effect of the medium was blocked by an IL-1 receptor antagonist. Moreover, exogenous IL-1β stimulated IL-6 mRNA expression in hepatocytes. IL-1β was present in the medium of non-parenchymal liver cells and increased with NE-treatment. These results suggest that NE released from sympathetic nerve terminals during stress can directly increase IL-6 mRNA expression in hepatocytes and indirectly through IL-1β production from non-parenchymal liver cells.View full abstractDownload PDF (2950K) -
Reiko Urade, Masahiko Kusunose, Tatsuya Moriyama, Takahiko Higasa, Mak ...2000 Volume 127 Issue 2 Pages 211-220
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe accumulation and degradation in the endoplasmic reticulum (ER) of a truncated ER-60 protease, from which the C-terminal 89 amino acid residues have been deleted (K 417 ochre), was examined. K 417 ochre overexpressed in COS-1 cells is not secreted into the medium, but accumulates as insoluble aggregates in non-ionic detergent without degradation in unusual clump membrane structures. K 417 ochre, stably expressed, forms soluble aggregates in non-ionic detergent and is distributed in the reticular structures of ER. Under these conditions, K 417 ochre is not secreted into the medium but is degraded with a half-life time of more than 8 h. Since K 417 ochre/C all S, in which all the Cys residues of K 417 ochre are replaced by Ser, also forms aggregates, an inter-disulfide bond appears unnecessary for aggregation. In both types of aggregates, Ig heavy chain binding protein, calnexin, glucose regulated protein 94, calreticulin, ERp 72, and protein disulfide isomerase are scarcely found. Since degradation of the stably expressed K 417 ochre was not inhibited by lactacystin, leupeptin, NH4Cl, or cytocharasin B, but was inhibited by N-acetyl-leucyl-leucyl-norleucinal, the self-aggregated abnormal protein in the lumen of ER is assumed to be degraded by an unknown protease system other than proteasome, lysosome or autophagy.View full abstractDownload PDF (5621K) -
Kikuo Ogata, Rie Ohno, Kazuo Terao, Kentaro Iwasaki, Yaeta Endo2000 Volume 127 Issue 2 Pages 221-231
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties and role in peptide elongation of ATPase intrinsic to rat liver ribosomes were investigated. (i) Rat liver 80S ribosomes showed high ATPase and GTPase activities, whereas the GTPase activity of EF-1α and EF-2 was very low. mRNA, aminoacyltRNA, and elongation factors alone enhanced ribosomal ATPase activity and in combination stimulated it additively or synergistically. The results suggest that these translational components induce positive conformational changes of 80S ribosomes by binding to different regions of ribosomes. Translation inhibitors, tetracyclin and fusidic acid, inhibited ribosomal ATPase with or without elongational components. (ii) Two ATPase inhibitors, AMP-P(NH)P and vanadate, did not inhibit GTPase activities of EF-1α and EF-2 assayed as uncoupled GTPase, but they did inhibit poly (U)-dependent polyphe synthesis of 80S ribosomes. (iii) Effects of AMP-P (NH) P and ATP on poly (U)-dependent polyphe synthesis at various concentrations of GTP were examined. ATP enhanced the activity of polyphe synthesis even at high concentrations of GTP, suggesting a specific role of ATP. At low concentrations of GTP, the extent of inhibition by AMP-P(NH)P was very low, probably owing to the prevention of the reduction of the GTP concentration. (iv) Vanadate inhibited the translocation reaction by high KCl-washed polysomes. These findings together indicate that ribosomal ATPase participates in peptide translation by inducing positive conformational changes of mammalian ribosomes, in addition to its role of chasing tRNA from the E site.View full abstractDownload PDF (1136K) -
Chiaki W. Nakagawa, Kenichiro Yamada, Norihiro Mutoh2000 Volume 127 Issue 2 Pages 233-238
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the induction of the catalase gene (ctt1+) of fission yeast Schizosaccharomyces pombe in response to several stresses by using mutants of transcription factors (Atf1 and Pap1) and a series of deletion mutants of the ctt1+ promoter region. A transcription factor, Atf 1, and its binding site are necessary for the induction of ctt1+ by osmotic stress, UV irradiation, and heat shock. Induction by menadione treatment, which produces superoxide anion, required element A, the region from -111 to -90 (numbered with the transcription start site as +1). The factor responsible for the induction of the gene by oxidative stress via element A was identified as the transcription factor Pap 1. We also found that Atf 1 is activated by menadione treatment in pap1 mutant cells, although it is not activated by menadione treatment in pap1+ cells. The activity of catalase is not increased in pap1 cells by several stresses, despite mRNA induction, suggesting that Papl plays some role in the expression of catalase activity.View full abstractDownload PDF (2792K) -
Uwe Bechtold, Jens T. Otterbach, Ralf Pasternack, Hans-Lothar Fuchsbau ...2000 Volume 127 Issue 2 Pages 239-245
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTransglutaminases (TGases, EC 2.3.2.13) have proved to be valuable enzymes for sitedirected protein coupling uia Nε-(γ-L-glutamyl)lysine bonds. Their use in conjugate synthesis would overcome many problems caused by chemical reagents. In this approach, we show for the first time that two proteins with different functionalities, namely soybean peroxidase and protein G, can be cross-linked by bacterial TGase with retention of their activities. Soybean peroxidase and protein G were chosen for the enzymic preparation of a bifunctional conjugate among a series of other TGase substrates detected by enzymic incorporation of small fluorescent or biotinylated molecules. The highest yields of conjugate were obtained with a 15-fold excess of peroxidase in phosphate buffer, pH 7.0. Size exclusion chromatography enabled both purification of the conjugates and recovery of the starting materials. Analysis of bifunctionality reveal-ed the coupling of protein G with an average of three peroxidase molecules.View full abstractDownload PDF (2805K) -
Yukio Sumi, Hisako Muramatsu, Ken-ichiro Hata, Minoru Ueda, Takashi Mu ...2000 Volume 127 Issue 2 Pages 247-251
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidkine (MK) is a heparin-binding growth/differentiation factor implicated in the control of development and repair of various tissues. To investigate the roles of MK in embryogenesis and in regulation of wound healing, we utilized a system of collagen gel contraction by dermal fibroblasts, which provides an in vitro model for studying fibroblast-collagen interactions important in various physiological and pathological phenomena. MK enhanced gel contraction 8-24 h after plating, and its effect was inhibited by anti-MK antibody. The effect was reduced after 48 h, while TGF-β continued to be active in the later stage. Morphologically, MK-treated fibroblasts tended to be elongated more frequently than control fibroblasts. The effect of MK in the early stage of gel contraction suggests a role of MR as a modulator of cell-matrix interactions.View full abstractDownload PDF (1648K) -
Takao Shishido, Mayumi Ohkawa, Akihiro Itoh, Takemi Enomoto, Yoshiyuki ...2000 Volume 127 Issue 2 Pages 253-261
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo monoclonal antibodies designated as 1F6 and 4B10 were obtained on screening for reactivities to CD 98-associated molecules by sandwich-type enzyme-linked immunosorbent assaying using hybridoma culture supernatants as the solid phase, cell lysates as an antigen source, and a mixture of biotinylated antibodies to CD98HC as a detector. Flow cytometric analysis with microspheres in combination with 1F6, 4B10, and anti-CD98HC also indicated the association of antibody-defined antigen(s) with CD98. 1F6 and 4B10, stained fibrillate components in fixed and permeated cells but were not reactive with unfixed live cells, suggesting that epitopes reside in the cytoskeleton-associated structure in the intracellular region. Two-color immunostaining followed by confocal microscopy revealed the colocalization of the antigen with CD98 at the cell-cell adhesion boundary of HeLa cells. 1F6 detected proteins with relative molecular masses of 33, 000 to 43, 000 on immunoblotting analysis involving cell lysates of human and rat cell lines. Analysis with a purified tropomyosin specimen from rabbit skeletal muscle demonstrated that 1F6 and 4B10 recognize tropomyosin. Two-dimensional gel electrophoresis followed by immunoblotting analysis revealed that 1F6 recognizes various tropomyosin isoforms. These results indicated that CD98 physically associates directly or indirectly with tropomyosin, and that this association is closely related to the cell-cell interaction.View full abstractDownload PDF (3773K) -
Keita Harada, Fumi Takahashi-Yanaga, Reiko Minakami, Sachio Morimoto, ...2000 Volume 127 Issue 2 Pages 263-268
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo explore the functional consequences of a deletion mutation of troponin T (ΔGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosinbinding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca2+ sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of ΔGlu160. These results provide strong evidence that the increased Ca2+ sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.View full abstractDownload PDF (2339K) -
Maosong Qi, Shinya Ikematsu, Keiko Ichihara-Tanaka, Sadatoshi Sakuma, ...2000 Volume 127 Issue 2 Pages 269-277
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, e. g. neuronal survival, carcinogenesis, and tissue repair. MK expression is detected mainly in the kidney in adult mice. In this study, we show that, at a dose that can induce recoverable renal damage and induce apoptosis, cisplatin (CDDP) transiently suppressed MK expression in mouse kidney. In vitro, CDDP suppressed MK expression and induced apoptosis in cultured G 401 cells, a Wilms' tumor cell line. Exogenous MK protein partially rescued G401 cells from CDDP-induced apoptosis. MK enhanced the expression of Bcl-2, but not that of Bcl-xL, in G401 cells in a dose-dependent manner, and it prevented the Bcl-2 reduction due to CDDP. Moreover, Bcl-2 expression in mouse kidney was also transiently suppressed by CDDP treatment, the expression profile being similar to that of MK. These results imply that MK exerts cytoprotective activity toward a damaging insult, presumably at least in part through enhancement of the expression of Bcl-2.View full abstractDownload PDF (4120K) -
Eiko Maeda-Nakai, Arata Ichiyama2000 Volume 127 Issue 2 Pages 279-287
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzymatic assay was developed for the spectrophotometric determination of glycolate in urine and plasma. Glycolate was first converted to glyoxylate with glycolate oxidase, and the glyoxylate formed was condensed with phenylhydrazine. The glyoxylate phenylhydrazone formed was then oxidized with K3Fe(CN)6 in the presence of excess phenylhydrazine, and A515 of the resulting 1, 5-diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxidase was carried out in 120-170mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris. The pyruvate formed from lactate was removed by subsequent brief incubation with alanine aminotransferase in the presence of L-glutamate, and α-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specificity of the assay relies principally on the substrate specificity of glycolate oxidase, and high sensitivity is provided by the high absorbance of 1, 5-diphenylformazan at 515-520 nm. Plasma was deproteinized with perchloric acid, and then neutralized with KOH. Plasma and urine samples were then incubated with _??_5mM phenythydrazine, and then treated with stearate-deactivated activated charcoal to remove endogenous keto and aldehyde acids as their phenylhydrazones. The normal plasma glycolate and urinary glycolate/creatinine ratio for adults determined by this method are _??_8 μM and _??_0.036, respectively.View full abstractDownload PDF (894K) -
Shigeki Taniguchi, Mieko Okinaka, Keiichiro Tanigawa, Ichitomo Miwa2000 Volume 127 Issue 2 Pages 289-295
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10mM glyceraldehyde was not completely inhibited by either 150 μM diazoxide (an opener of ATP-sensitive K+ channels) or 5 μM nitrendipine (an L-type Ca2+-channel blocker), whereas the stimulation of insulin secretion by 20mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 μM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myoinositol phosphate production by 100 μM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca2+ through voltage-dependent Ca2+ channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.View full abstractDownload PDF (772K) -
Masahiko Kato, Takashi Nonaka, Masatoshi Maki, Hidehiko Kikuchi, Shino ...2000 Volume 127 Issue 2 Pages 297-305
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously reported the activation of procalpain μ (precursor for low-calciumrequiring calpain) in apoptotic cells using a cleavage-site-directed antibody spedcific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.View full abstractDownload PDF (3873K) -
Tomofumi Fujino, Tomohiro Tada, Toshiki Hosaka, Masatoshi Beppu, Kiyom ...2000 Volume 127 Issue 2 Pages 307-313
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [3H] DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [3H] DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [3H] DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.View full abstractDownload PDF (2330K) -
Yaw-Kuen Li, Hsin-Jan Yao, I-Hong Pan2000 Volume 127 Issue 2 Pages 315-320
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe catalytic mechanism of the β-xylosidase purified from the culture filtrate of Trichoderma koningii G-39 was investigated. By NMR spectroscopy, the stereochemistry of the enzyme catalyzing the hydrolysis of 2, 4-dinitrophenyl and p-nitrophenyl-β-xylosides was found unequivocally to involve retention of the anomeric configuration. Based on the kcat values of a series of arylxylosides with leaving group pKas in the range of 4-10, an extended Bronsted plot was constructed with a slope (β1g) near zero. Enzymatic hydrolysis of aryl-β-D-xylosides in acetate buffer (pH 4.0) containing 3 or 5% methanol showed a constant product ratio (methylxyloside/xylose), indicating the presence of a common intermediate, probably the xylosyl-enzyme intermediate. In the presence of DTT, the kcat values of p-cyanophenyl-β-D-xylopyranoside and p-nitrophenyl-β-D-xylopyranoside increased greatly. A two-step mechanism involving the formation and breakdown of the xylosyl-enzyme intermediate was therefore proposed. The rate-limiting step is the breakdown of the intermediate. The secondary deuterium kinetic isotope effect (kH/kD) measured for 2, 4-dinitrophenyl-β-D-xyloside was 1.02±0.01, suggesting that the transition state for breakdown of the xylosyl-enzyme intermediate is SN2-like.View full abstractDownload PDF (1007K) -
Shinobu Tsuchida, Shigeru Yanagi, Ryoko Inatome, Junyi Ding, Patrice H ...2000 Volume 127 Issue 2 Pages 321-327
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSyk protein-tyrosine kinase (PTK) has been implicated in a variety of hematopoietic cell responses including immunoreceptor signaling. However, so far, there has been no evidence of the expression of Syk or Syk-related PTK in non-hematopoietic tissues. In this study, we have purified from blood cell-depleted rat liver a 72-kDa cytoplasmic PTK which shows cross-reactivity with anti-Syk antibody. Partial amino acid sequence analysis revealed that this 72-kDa PTK is identical to Syk. Immunohistochemical and RT PCR analyses demonstrated that Syk is expressed in human hepatocytes and two rat liverderived cell lines, JTC-27 and RLC-16. Furthermore, Syk is significantly tyrosine-phosphorylated in response to angiotensin II in JTC-27 cells, and angiotensin II-induced MAP kinase activation is blocked by the treatment of cells with a Syk-selective inhibitor, piceatannol. These results suggest that Syk plays an important role in signaling events of hepatocytes, such as signaling steps leading to MAP kinase activation by G-proteincoupled receptors. This is the first report of the expression of Syk in non-hematopoietic tissue.View full abstractDownload PDF (3086K) -
Takashi Matsumoto, Yukio Morimoto, Naoki Shibata, Takashi Kinebuchi, N ...2000 Volume 127 Issue 2 Pages 329-335
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe single-stranded DNA (ssDNA) binding protein from Escherichia coli (EcoSSB) plays a central role in DNA replication, recombination, and repair. The tertiary structure of EcoSSB was determined at 2.2 Å resolution. This is rather higher resolution than previously reported. Crystals were grown from the homogeneous intact protein but the EcoSSB tetramer in the crystals contains truncated subunits lacking a part of the C-terminal. The structure determined includes biologically important flexible loops and C-terminal regions, and revealed the existence of concavities. These concavities include the residues important for ssDNA binding. An ssDNA can be fitted on the concavities and further stabilized through interactions with the loops forming flexible lids. It seems likely to play a central role in the binding of ssDNA.View full abstractDownload PDF (3448K) -
Shinya Oue, Akihiro Okamoto, Takato Yano, Hiroyuki Kagamiyama2000 Volume 127 Issue 2 Pages 337-343
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mutant Escherichia coil aspartate aminotransferase with 17 amino acid substitutions (ATB 17), previously created by directed evolution, shows increased activity for β-branched amino acids and decreased activity for the native substrates, aspartate and glutamate. A new mutant (ATBSN) was generated by changing two of the 17 mutated residues back to the original ones. ATBSN recovered the activities for aspartate and glutamate to the level of the wild-type enzyme while maintaining the enhanced activity of ATB 17 for the other amino acid substrates. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, also returned to the original state. ATBSN shows significantly increased affinity for substrate analogs including succinate and glutarate, analogs of aspartate and glutamate, respectively. Hence, we could cocrystallize ATBSN with succinate or glutarate, and the structures show how the enzyme can bind two kinds of dicarboxylic substrates with different chain lengths. The present results may also provide an insight into the long-standing controversies regarding the mode of binding of glutamate to the wild-type enzyme.View full abstractDownload PDF (773K) -
Kunishige Kataoka, Hiroshi Furusawa, Kohichi Takagi, Kazuya Yamaguchi, ...2000 Volume 127 Issue 2 Pages 345-350
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme. Moreover, two conserved noncoordinate residues, Asp 98 and His 255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR. The Asp 98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His 255 residue hydrogen-bonded to Asp 98 through the water molecule was replaced with Ala, Lys, or Arg. The catalytic rate constants of all mutants were decreased to 0.4-2% of those of the recombinant enzyme, and the apparent Km values for nitrite were greatly increased in the Asp 98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp 98 and His 255 are involved not only in the catalytic reaction but also in the substrate anchoring.View full abstractDownload PDF (2292K)
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