巻号一覧
121 巻, 1 号
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Tomohiko Toyoda, Takeshi Sekiguchi, Akio Takenaka1997 年 121 巻 1 号 p. 1-4
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーLipoamide dehydrogenase, which is a common component of a-keto acid dehydrogenase complexes, has been highly purified from yeast (Saccharornyces cerevisiae) toreveal its structure at higher resolution. New crystals obtained by a desalting method exhibited diffraction beyond 2. 5 Å resolution. The cell dimensions are a=97.1, b=158.7, and c=67.9Å, and the space group is P212121. There isa dimeric enzyme in the asymmetric unit. The crystal structure was solved by means of the molecular-replacement technique and refined in a preliminary manner.抄録全体を表示PDF形式でダウンロード (1295K) -
Kenji Ikeda, Hiroshi Wachi, Yoshiyuki Seyama, Shingo Tajima1997 年 121 巻 1 号 p. 5-7
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーTreatment with 80 nM monensin for 48 h resulted in impairment of tropoelastin secretion during the initial 6 h and subsequent reduction of tropoelastin synthesis from 18 to 48 h to one-tenth. The steady state level of tropoelastin mRNA started to decrease at 6 h and reached one-fifth of the control level by 48 h. A pulse-chase experiment after 24 h monensin treatment demonstrated that half of the accumulated tropoelastin in the cells was rapidly degraded within 120 min. These results indicate that the marked reduction in tropoelastin synthesis from 6 to 48h treatment may be caused by both a reduction in the tropoelastin mRNA level and accelerated intracellular degradation of tropoelastin. Thus, monensin modulates tropoelastin expression through pretranslational and posttranslational mecha-nisms.抄録全体を表示PDF形式でダウンロード (1188K) -
Soh Yamazaki, Satoshi Harashima, Masao Sakaguchi, Katsuyoshi Mihara1997 年 121 巻 1 号 p. 8-14
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーCoatomer, the cytosolic protein complex, consists of seven subunits (α, β, β, γ, δ, ε andξ-COP) and is involved in vesicle trafficking early in the secretory pathway in collaboration with a 20 kDa GTP-binding protein (ARF). In the present study, we have identified a yeast gene which encodes a protein having 39% amino acid sequence identity with bovine ξ-COP. This gene (YZC1 for yeast zetaCOP) is essential for vegetative growth and the growth defect of Δyzel cells was restored by bovine ξ-COP cDNA. We isolated a temperature-sensitive mutant of YZC1 (yzclts) and examined its capacity for both the ER-to-Golgi transport and the double lysine motif (KKXX)-mediated retrograde transport from Golgi to ER. Atnon-permissive temperature, the yzelte cells exhibited a weak defect in the anterograde transport, but a strong defect in the retrograde vesicle transport. We conclude that Yzclp is a yeast homologue of mammalian ξ-COP and participates mainly in the Golgi-to-ER retrograde transport.抄録全体を表示PDF形式でダウンロード (3744K) -
Jin-Chul Kim, Soo Kyoung Bae, Jong-Duk Kim1997 年 121 巻 1 号 p. 15-19
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーTemperature-sensitive drug release was examined using liposomes mixed with a copoly-mer of N-isopropylacrylamide (NIPAM) and acrylic acid [P(NIPAM-AA)] i. e., thermally responsive liposomes. P(NIPAM-AA) copolymers with transition temperatures of about 30, 33, 37, and 43°C were synthesized by copolymerizing NIPAM and acrylic acid. Thermally responsive liposomes were prepared by mixing hydrophobically modified PNIPAM, or P(NIPAM-AA) with various liposomes, composed of egg phosphatidylcholine (PC), dimy-ristoylphosphatidylcholine (DMPC)/dipalmitoylphosphatidylcholine (DPPC) mixture (5 : 5, w/w), DPPC, or distearoylphosphatidylcholine (DSPC). The release of a fluorescent marker, calcein, from liposomes was monitored by injecting the liposomal suspension at 17°C into phosphate-buffered saline (PBS, pH 7.4) preadjusted to a temperature ranging from 20 to 46°C. For liposomes of egg PC and DSPC, which do not undergo a phase transition during the temperature jump (17→20-46°C), the release temperature of the liposomes increased as the content of acrylic acid in the copolymers increased. The interaction between copolymer and lipid may induce the release of calcein at LCST of the copolymer. For DPPC liposomes, the release patterns were similar to those of egg PC and DSPC liposomes at 20-36°C, where the phase transition of the liposomal membrane did not occur, while at 36-46°C, where the phase transition of liposomal membrane occurred, the degree of release was almost the same. For DMPC/DPPC (5 : 5, w/w) liposomes, where the transition occurred below those of PNIPAMs, equally enhanced releases were observed as compared with PNIPAMs, even below the LCSTs of PNIPAMs. Thus, regardless of the occurrence of the transition of PNIPAMs, phase transition of DMPC/DPPC liposomes controlled the release of calcein.抄録全体を表示PDF形式でダウンロード (566K) -
Takao Matsuba, Mayumi Yano, Norio Abiru, Hirofumi Takino, Shoichi Akaz ...1997 年 121 巻 1 号 p. 20-24
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーDetection of serum autoantibodies to glutamic acid decarboxylase (GAD) is a new method to differentiate insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). We established a transformed mouse myeloma cell line, SPG14, which expresses recombinant human GAD65, a major isomer of 65 kDa, inside the cells. GAD65 was partially purified by affinity chromatography using the mouse anti-GAD antibody (Ab). We also established a sandwich ELISA for anti-GAD Ab of the IgG class using GAD65 for coating and the anti-human IgG for detection and examined 54 sera of the IDDM patients and 45 sera of normal individuals. When the mean +2 SD of the color development of the sera of normal individuals was used as a cut-off level, 59. 2%of patients with IDDM were positive. This indicates that the ELISA was effective to differentiate IDDM and NIDDM. The result also indicates that the autoantibody to GAD does not dissociate from the recombinant GAD rapidly, even when unbound anti-GAD Ab was fully removed. Byusing a perfusion culture system, we obtained as many as 4.2 X 1010 SPG14 cells, from which 5 mg of GAD65 could be obtained; this is sufficient for 5, 000 assays. This system could be clinically useful for large-scale diagnosis of IDDM.抄録全体を表示PDF形式でダウンロード (1552K) -
Osamu Soga, Hiroshi Kinoshita, Mitsuyoshi Ueda, Atsuo Tanaka1997 年 121 巻 1 号 p. 25-28
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーHeme is supposed to be transported into peroxisomes to form peroxisomal catalase[EC 1. 11. 1. 6] in harmony with proliferation of the organelle because of the absence of the heme synthetic pathway in peroxisomes. We tried to understand the transport mechanism of peroxisomal catalase through the peroxisomal membrane from the aspects of a cofactor, heme, by measuring cellular and subcellular heme contents with the pyridine hemochrome method, independent of measuring the catalase activity. n-Alkane-grown Candida tropi-calis cells, in which peroxisomes develop profusely, contained a larger amount of heme than the glucose-grown cells, and the increase well matched that of the catalase activity. The results of subcellular fractionation of the n-alkane-grown cells showed that, in peroxisomes, the catalase subunit and heme existed in a molar ratio of 1:1, indicating that excessively transported catalase subunit proteins or heme could not be present in perox-isomes and they formed a tetramer having four molecules of heme. From this quantification of peroxisomal heme, it was strongly suggested that the amounts of the catalase subunit protein and heme transported into peroxisomes should be stoichiometrically regulated at the step of their synthesis or/and transport.抄録全体を表示PDF形式でダウンロード (484K) -
Jun Sakanoue, Kazuhiko Ichikawa, Yasutomo Nomura, Mamoru Tamura1997 年 121 巻 1 号 p. 29-37
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーTo examine the feasibility of optical monitoring of cellular energy states with tissue-trans-parent near-infrared (NIR) light, the absorption and fluorescence characteristics of Rhodamine 800 in isolated rat liver mitochondria and hepatocytes were investigated. When the dye was incubated with isolated mitochondria, alarge red shift of the absorption spectra and quenching of the fluorescence intensity were observed. The absorbance difference at 730 minus 685 nm, or at 730 minus 800 nm, and the fluorescence intensity measured at 692 nm varied linearly with the mitochondrial membrane potential. The spectral changes observed could beexplained in terms of the potential-dependent uptake of the dye from the buffer solution into the mitochondrial matrix. The respiration control ratio and oxygenconsumption rate were not affected by the addition of Rhodamine 800 at concentrations lower than 5 μM, which was the concentration range mostly employed throughout the present study. In a suspension of hepatocytes, the red shift and fluorescence quenching of Rhodamine 800 characteristic of energized mitochondria were also observed, and these changed to those of the buffer solution with the addition of an uncoupler under normoxia. At the early stage of anoxia, within about 5 min, when cytochrome oxidase was completely reduced, hepatocytes were concluded to be in the fully energized state, since the optical characteristics of Rhodamine 800 were the same as those of energized mitochondria. On the basis of these in vitro data, Rhodamine 800 is concluded to be a possible NIR-active contrast agent, that can be used to monitor the energy states of living tissues, in addition to the tissue oxygenation states, by the use of near-infrared spectrophotometry (NIRS) without harmful effects.抄録全体を表示PDF形式でダウンロード (1023K) -
Tetsuji Noda, Fumio Ammo1997 年 121 巻 1 号 p. 38-46
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーNitric oxide synthase (NOS) activities were compared in a macrophage-like cell line, RAW 264. 7 cells, treated with bacterial lipopolysaccharide (LPS) alone or with LPS and inter-feron-γ(IFN-γ). An about 5-6-fold higher amount of NO2-originating from the nitric oxide radical (•NO) was produced in the culture supernatant of macrophages treated with LPS+ IFNγ than with LPS alone, depending on both the time of incubation and the dose of LPS. However, the difference in the amounts of iNOS protein in these macrophage extracts was much greater than that inthe amount of released NO2-. To estimate the NOS activity after induction of NOS at 37°C for 8h, we examined both intact cells and cell extracts. The cells were washed and re-incubated in Hank's balanced salt solution (HBSS) containing 1 mM L-arginine and 100 μM carboxy-PTIO, which enabled us to detect trace amountsof NO by means of rapid reaction of carboxy PTIO with NO to form-NO2, which was finally converted to NO2-. The results showed an about 7-fold difference betweenthe macrophages pretreated with LPS alone and those with LPS +IFN-γ. Using the extracts, the NOS activity was assayed with L-[ U-14C] arginine as a substrate for NOS in vitro, and the results again revealed an about 7-fold difference between the two types of cell extracts. A kinetic study of the NOS activity by means of in vitro assay suggested that there was little difference in Km value for L-arginine between these two iNOSs. However, it revealed two apparent Kms for, β-NADPH, a co-factor of NOS; one was about 0. 4 μM, which was common to these two iNOSs, and the other was about 1. 5 and 25 μM for LPS-induced NOS and LPS +IFN-γ-induced NOS, respectively. The cellular concentration of γ-NADPH was around 14 μM in both LPS-and LPS IFN-β-treated macrophages. These results suggest that there is some heterogeneity of iNOSs induced in macrophages with LPS alone and with LPS +IFN-β, and the heterogeneity seems to be due at least in part to the requirement for β-NADPH.抄録全体を表示PDF形式でダウンロード (2743K) -
Hajime Honda, Nobuyuki Tagami, Kuniyuki Hatori, Koichiro Matsuno1997 年 121 巻 1 号 p. 47-49
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーTroponin-tropomyosin complex from skeletal muscles was observed to regulate sliding movement of actin filaments on myosin molecules in a manner independent of their ATPase activity. When actin molecules were crosslinked with DSS (disuccinimidyl suberate), the myosin ATPase activity in the presence of the modified actin filaments complexed with both troponin and tropomyosin was only 10% less than that in the case of unmodified actin, and the ATPase activation was independent of calcium ions. In contrast, the sliding velocity of the modified actin filaments on myosin molecules decreased to zero below pCa 6.5. The present results indicate that troponin-tropomyosin complex regulates contractile move-ment of actomyosin systems through direct alternation of a mechanochemical property of the thinfilaments, not through a decrease in the ATPase activity of the myosin molecules.抄録全体を表示PDF形式でダウンロード (394K) -
Kanako Katai, Hiroko Segawa, Hiromi Haga, Kyoko Morita, Hidekazu Arai, ...1997 年 121 巻 1 号 p. 50-55
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーAlteration of the dietary intake of phosphate (Pi) leads to rapid changes in renal Pi transport activity. The present study, examined the underlying cellular mechanisms of the rapid regulation, with special reference to renal P, cotransporter. Rats were fed either a low-Pi (0. 02%) diet (CLP rats), the low-Pi diet followed by a high-P, (1. 2%) diet (AHP rats), or a normal (0. 6%) diet (control rats). Nat-dependent Pi transport activity in the brush border membrane was significantly increased in CLP rats compared with control rats, and this activity decreased rapidly within 2 h after the change of diet in AHP rats. Kinetic analysis of Pi transport in the AHP rats indicated that the reduction was accompanied by a decrease in the apparent Vmax, for Na+-dependent Pi uptake. Northern blot analysis showed no difference in the abundance of NaPi-2 mRNA of the kidney between AHP and CLP rats. In contrast, Western blot analysis of renal brush border membrane proteins of AHP rats indicated a significant decrease in the abundance of NaPi-2 protein as compared with CLP rats. Immunoreactive signals for NaPi-2 were detected in lysosomal fractions of AHP and CLP rats. Immunohistochemical analysis showed that, NaPi-2 immunoreactivity in AHP rats was largely reduced in the apical membrane of the proximal tubular epithelial cells. Neither cycloheximide nor actinomycin D affected high-Pi-induced reduction of NaPi-2 protein in the brush border membrane of AHP rats, indicating that de novo protein synthesis of an unidentified regulator protein was not involved in the mechanism of this reduction. In contrast, treatment with colchicine, which disrupts microtubulers, abolished the effect of high-Pi diet on NaP, -2 expression. These results suggested that rapid en-docytotic internalization of NaP, -2 may occur specificallyin the brush border membrane following an acute increase in dietary Pi intake.抄録全体を表示PDF形式でダウンロード (4926K) -
Tsuyoshi Katoh, Fumi Morita1997 年 121 巻 1 号 p. 56-62
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーPorcine aorta smooth muscle myosin contains two essential light chain (LC17) isoforms and the light chain was replaced with one of the LC17 isoforms, rabbit skeletal muscle myosin alkali light chain 2 (A2), or scallop striated muscle myosinessential light chain (SHLC). The myosin containing either an LC17 isoform or A2showed phosphorylation-dependent properties in the monomer conformation, filament formation, ATPase activities, and superprecipitation, behaving in essentially the same way as native myosin. On the other hand, the replacement of LC17 with SHLC destabilized the 10S conformation and the myosin was predominantly filamentous under physiological conditions, irrespective of the phos-phorylation state. This myosin containing dephosphorylated regulatory light chain showed higher actin-activated ATPase activity than native dephosphorylated myosin and was further activated by Ca2+, resulting in a decrease of phosphorylation-dependent regulation. Superprecipitation for the myosin was observed only when the regulatory light chain was phosphorylated. Superprecipitation for myosin containing SHLC was significantly slow in the absence of Ca2+ in comparison with that for myosin containing LC17, and was further activated by the presence of Ca2+. On the basis of the differences in amino acid sequences of these essential light chains, it appears that the N-terminal domain of LC17 may be implicated in these phosphorylation-dependent properties of smooth muscle myosin.抄録全体を表示PDF形式でダウンロード (2009K) -
Tomomitsu Hatakeyama, Yoko Miyamoto, Haruna Nagatomo, Imure Sallay, No ...1997 年 121 巻 1 号 p. 63-67
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe carbohydrate-binding properties of the hemolytic lectin CEL-III from the Holothuroidea Cucumaria echinata were studied using the microplate assay system which we have recently developed [Hatakeyama et al. (1996) Anal. Biochem. 237, 188-192]. When the binding of CEL-III to lactose covalently immobilized on a microplate was examined using colloidal gold solution, the binding was detected with as little as 1 μg/ml protein. Affinity of several carbohydrates to CEL-III was assessed by means of an inhibition experiment using the lactose-coated plate and it was found that N-acetylgalactosamine has the highest affinity for CEL-III, followed by lactose and lactulose. Examination of the binding of CEL-III to the lactose-coated plate at various pH values and temperatures revealed that the affinity is higher in the acidic pH region and at lower temperatures. From the Ca2+- dependence profile for the binding of CEL-III to the lactose-coated plate, the apparent dissociation constant for Ca2+ was estimated to be 2.3mM. These results suggested that the carbohydrate-binding properties of CEL-III are closely related to its hemolytic activity, although an additional interaction between the protein and the lipid bilayer, which is enhanced in the alkaline pH region, also seems to be necessary for its hemolytic action.抄録全体を表示PDF形式でダウンロード (560K) -
Takao Kawakami, Masaharu Kamo, Keiji Takamoto, Kenji Miyazaki, Lu-Ping ...1997 年 121 巻 1 号 p. 68-76
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーPeptide bond cleavages by vapors composed of various from aqueous solutions of perfluoric acid were studied using synthetic peptides and proteins, and specific conditions were established for peptide bond cleavages including a novel cleavage of the glycyl-threonine bond. The peptide bonds on the aminosides of serine residues were cleaved by exposure to a vapor of 75% aqueous heptafluorobutyric acid at 30 or 50°C for 24h. Glycyl-threonine peptide bonds were cleaved with vapors of various concentrations (5, 75, and 90%) of heptafluorobutyric acid at 30-40°C for 24h. The peptide bonds on the carboxylsides of aspartic acid residues were cleaved by exposure to a vapor of 0.2% heptafluorobutyric acid at 90°C for 4 to 24h. The same vapor cleaved aspartyl-proline bonds under milder conditions such as at 60°C for 16h, under which the other aspartyl bonds were uncleaved. These specific chemical cleavages were applied to several proteins including newly characterized proteins.抄録全体を表示PDF形式でダウンロード (4912K) -
Takuro Yaoi, Kentaro Miyazaki, Tairo Oshima1997 年 121 巻 1 号 p. 77-81
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe substrate-binding sites of NADP-dependent isocitrate dehydrogenase and NAD-depen-dent 3-isopropylmalate dehydrogenase from Thermus thermophilus were analyzed by site-directed mutagenesis. Ser97 and Asn99 of isocitrate dehydrogenase were identified to be involved in the isocitrate recognition. In 3-isopropylmalate dehydrogenase, the corresponding residues, Leu90 and Leu91, appear to recognize the substrate by forming a hydrophobic pocket. Double mutation of Asp78 and Glu87 revealed that negative charge of these residues plays a crucial role in discriminating isopropylmalate from isocitrate.抄録全体を表示PDF形式でダウンロード (1126K) -
Hee-Jung Kim, Yoshiaki Tamanoue, Gwang-Ho Jeohn, Akihiro Iwamatsu, Aki ...1997 年 121 巻 1 号 p. 82-88
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーAn extracellular metalloprotease was purified from the culture supernatant of Pseudomonas fluorescens strain KT1 to apparent homogeneity and shown to consist of a single polypeptide chain (Mr, 46, 000-47, 000). The enzyme was strongly inhibited by chelating agents such as EDTA and o-phenanthroline, and activated by certain detergents. Among the peptidyl 4-methylcoumaryl-7-amide (MCA) substrates examined, t-butyloxycarbonyl-Arg-Val-Arg-Arg-MCA was the best one. With this substrate, the enzyme exhibited a pH optimum of around pH 5.5 in the absence of Co2+ ions, whereas it showed two different pH optima (at pHs around 5.5 and 8-9) in the presence of Co2+ ions due to remarkable activation by Co2+ ions in the alkaline pH range. On the other hand, a single broad pH optimum of around 6 to 8 was obtained with some peptides in both the presence and absence of Co2+ ions, and no activation by Co2+ was observed. The enzyme showed trypsin-like specificity, preferentially cleaving certain arginyl peptide bonds, and hydrolyzed the basic protein, histone, most rapidly among various proteins examined. Partial amino acid sequence analysis revealed that the enzyme is highly homologous with proteases of the serralysin family, a group of zinc metalloproteases.抄録全体を表示PDF形式でダウンロード (878K) -
Kenji Sakoda, Asuka Suzuki, Shigeki Arai, Masayoshi Imagawa, Jun-ichi ...1997 年 121 巻 1 号 p. 89-94
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリー1α, 25-Dihydroxyvitamin D3, [1, 25-(OH)2, D3], the active form of vitamin D3, has been thought to be a multifunctional agent. In order to discover novel roles of 1, 25-(OH)2 D3, we have been looking for new genes that are regulated by 1, 25-(OH)2 D3. Because the actions of 1, 25-(OH)2D3 are mediated through the vitamin D receptor (VDR), that is a DNA binding transcription factor, vitamin D regulated genes should have VDR binding sites in their regulatory regions. In this paper, we describe a novel vitamin D response element (VDRE)- containing sequence, clone 3, which was isolated through binding to VDR. DNA sequence analysis of clone 3 did not reveal any significant similarity with sequences reported previously. Clone 3 had two regions consisting of a direct repeated sequence of AGTTCA motifs, both of which bound to VDR independently. Whereas each direct repeat sequence alone could not mediate transcriptional activation efficiently, with their co-existence there was a strong response to 1, 25-(OH)2 D3, indicating that these two direct repeated sequences act cooperatively.抄録全体を表示PDF形式でダウンロード (2527K) -
Ken Yanagisawa, Yoshiyuki Naito, Kenji Kuroiwa, Takao Arai, Yusuke Fur ...1997 年 121 巻 1 号 p. 95-103
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe ST2 gene, which is specifically induced by growth stimulation, encodes interleukin-1 receptor-related proteins. Using the RT-PCR method, we found that the ST2 gene was broadly expressed in hematopoietic cell lines. It was also expressed specifically in helper T cell lines among lymphocytic cell lines. We analyzed the expression of ST2 in mouse helper T cell subsets with Northern blotting analysis. Mouse Th1 cell lines so far studied did not express ST2 mRNAs. On the other hand, one of the Th2 cell lines, D10, expressed ST2L (transmembrane form) without stimulation, while co-stimulation by PMA and A23187 induced ST2 (soluble form) mRNA. These results suggest that the ST2 gene is involved in the regulation of the immune system. IL-1α, IL-1β, and receptor antagonist did not bind to ST2L protein, which prompted us to search for the specific ligand of ST2. The recombinant human ST2 protein was purified and labeled with FITC. The labeled human ST2 protein bound with myeloma-derived RPMI8226 cells among the various B-cell lines, indicating possible involvement of ST2 in T-cell/B-cell interaction.抄録全体を表示PDF形式でダウンロード (4796K) -
Masahiro Ariki, Osamu Tanabe, Hirofumi Usui, Hideyuki Hayashi, Rintaro ...1997 年 121 巻 1 号 p. 104-111
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーc-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+• ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNE_??_TAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, _??_TPENPTEPVNTSAGH_??_GVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.抄録全体を表示PDF形式でダウンロード (2326K) -
Inger M. Janson, Bo Ek, Pia Ek1997 年 121 巻 1 号 p. 112-117
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーSeveral proteins in the mammalian endoplasmic reticulum are substrates for protein kinases. Many unidentified phosphoproteins from this compartment are described in the literature, and this prompted us to try to identify at least the more dominant ones. When solubilized bovine and murine microsomes were phosphorylated with protein kinase CK2 and [32P] ATP and separated on SDS-PAGE, the corresponding autoradiogram showed three dominant 32P-labeled proteins. These three [32P]phosphoproteins were identified as calcium-binding proteins (CaBP) 1, 2, and 4 after purification on a MonoQ column followed by SDS-PAGE, proteolytic cleavage and subsequent amino acid sequencing of the purified 32P-labeled peptides. All three were also phosphorylated by an endogenous kinase, found by us to be of the CK2 type. This kinase phosphorylated CaBP1 N-terminally at serine 427. Of the three proteins, only CaBP4 was previously known to be a substrate of CK2. The newly identified substrates CaBP 1 and 2 are members of the thioredoxin family and have a signal tetrapeptide in the C-terminal of the protein for retention in the ER. Serines and/or threonines in the C-terminal were phosphorylated in CaBP1 when the endogenous CK2 was used as protein kinase. A protein with the same molecular mass as CaBP1 on SDS-PAGE was phosphorylated when intact hepatocytes were grown in the presence of [32P] phosphate. The in vitro phosphorylation with protein kinase CK2 can be used as a specific and sensitive method for identification of CaBP1, 2, and 4 in microsomes.抄録全体を表示PDF形式でダウンロード (3214K) -
Young-Nam Park, Jun-ichi Aikawa, Makoto Nishiyama, Sueharu Horinouchi, ...1997 年 121 巻 1 号 p. 118-121
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーReplacement of Trp39 of Rhizomucor pusillus pepsin (RMPP) by Asn or Cys resulted in a marked decrease in the milk-clotting and proteolytic activities. Kinetic analysis with chromogenic synthetic oligopeptides as substrates revealed that the mutations caused marked changes in the kcat, value, but only slight changes in the Km value. Similar enzymatic properties were observed in mutants of Tyr75, which was shown to have a role in enhancing the catalytic activity. Both Tyr75Asn and Trp39Asn mutants rapidly lost the activity at high temperatures due to autocatalytic digestion at two sites. The structures of several aspartic proteinases including RMPP, as revealed by X-ray crystallographic studies, showed that Trp39 occupies a position close to Tyr75 and the N8 atom of Trp39 within hydrogen-bonding distance of the hydroxyl side chain of Tyr75. These observations suggest that Trp39 plays a role in maintaining Tyr75 in the correct orientation in aspartic proteinases, including RMPP.抄録全体を表示PDF形式でダウンロード (1536K) -
Tomokazu Aoki, Tatsuro Koike, Toru Nakano, Keiichi Shibahara, Shigeru ...1997 年 121 巻 1 号 p. 122-127
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーWe have found that expression of the Bip (immunoglobulin heavy chain binding protein)/GRP78 (glucose regulated protein 78) gene is markedly enhanced specifically among the heat shock protein (HSP) 70 gene family during the neuronal cell death of PC12 (22a) cells, that is induced by removal of nerve growth factor (NGF) and blocked by a transcription inhibitor, actinomycin D. The Bip mRNA induction is suppressed when the NGF-deprivation-dependent cell death of PC12 (22a) cells is inhibited by cAMP, cycloheximide or high K+. The Ca2+ ionophore, A23187, caused neuronal cell death accompanied by up-regulation of Bip, HSP90, and HSP70 mRNAs. In addition, a chelator of intracellular Ca2+ (BAPTA) elevated Bip mRNA and induced cell death in a low Ca2+ medium. Alterations of intracellular calcium homeostasis thus appear to induce Bip mRNA expression as well as apoptosis in PC12 (22a) cells. However, release of Ca2+ from intracellular stores by thapsigargin induced Bip mRNA expression but not cell death, indicating that Bip mRNA induction is not sufficient for neuronal death. Induction of Bip mRNA in association with apoptosis was also observed for NGF-deprived sympathetic ganglion cells in primary culture. These lines of evidence suggest that selective induction of Bip mRNA may play an important role in the programmed cell death of neurons deprived of neurotrophic factors and could be a landmark of the neuronal programmed cell death.抄録全体を表示PDF形式でダウンロード (3798K) -
Yoshihisa Hagihara, Kei-ichi Enjyoji, Takeshi Ômasa, Yoshio Kata ...1997 年 121 巻 1 号 p. 128-137
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーIn order to elucidate the mechanism of binding of β2-glycoprotein I (β2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of β2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human β2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i. e., the peptide bond between Lys 77 and Thr 78. The conformation of the “nicke” Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.抄録全体を表示PDF形式でダウンロード (1894K) -
Takashi Matsumoto, Nobuhiko Nakashima, Kenji Takase, Hirohiko Hirochik ...1997 年 121 巻 1 号 p. 138-144
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーA site-specific mutation study was performed on the C-terminal domain, containing a cloned DNA binding region, of the human papillomavirus type11 (HPV11) E2 protein to determine the specific properties of residues directly involved in the DNA binding. The effect of a point mutations on the DNA binding was assessed by means of a gel mobility shift assay. The mutagenesis was concentrated on the residues in the third helix from the Nterminal, that is known as the “recognition helix, ” in the crystal structure of the bovine papillomavirus (BPV) E2 protein. Most point mutations caused a great decrease in the DNA binding activity. The leucine repeat in the DNA binding region was proved not to be a leucine prerequisite, as the leucines could be substituted by valine without significant loss of the DNA binding ability. Substitution of Leu for Glu caused a significant decrease in the DNA binding, indicating that the hydrophobicity of the residue at this position is important. The results suggest that the individual contribution of each amino acid residue in the DNA binding region is essential for the DNA binding.抄録全体を表示PDF形式でダウンロード (4308K) -
Hiroshi Tachikui, Apala Farhat Navet, Masayuki Ozawa1997 年 121 巻 1 号 p. 145-149
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーReticulocalbin (RCN) is a member of the EF-hand Ca2+-binding protein family and is a luminal protein of the endoplasmic reticulum (ER) with a molecular weight of 44, 000 [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Although RCN has six repeats of a domain containing an EF-hand motif, the varying degrees of divergence of the amino acid sequences of these domains from the EF-hand consensus sequences suggested that some domains might have lost their Ca2+-binding capability and adopted new functions. To identify the domains involved in Ca2+-binding, discrete domains of RCN were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system. 45Ca2+ blot analysis of the resultant fusion proteins revealed that the first, fourth, fifth, and sixth domains bind Ca2+, however, the second and third ones do not. The fusion proteins containing all six domains, and the first and second domains, respectively, showed Ca2+-dependent increases in their electrophoretic mobilities, suggesting that Ca2+ induces a conformational change in reticulocalbin.抄録全体を表示PDF形式でダウンロード (1459K) -
Richard Jennemann, Bernhard L. Bauer, Herbert Wiegandt1997 年 121 巻 1 号 p. 150-154
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーExposure of glycosphingolipids to air irreversibly destroys the integrity of these lipids within a few hours. It was established that among the natural constituents of air, ozone, at commonly observed daytime levels, is responsible for the observed degradation. As one product of the reaction of glycosphingolipids with air, an aldehydic fragment containing the carbohydrate moiety was identified. This aldehydic fragment was identical to the one obtained by classical glycosphingolipid ozonolysis. Identical with the latter, the air-induced product is further fragmented by mild alkali treatment with concomitant liberation of the free reducing oligosaccharide. As a consequence of the alteration of glycosphin-golipids by air, it was shown that the accuracy of methods of analysis of these glycocon-jugates that depend on their physico-chemical integrity, e. g., by ticimmune overlay, is severely influenced by their prior exposure to the atmosphere.抄録全体を表示PDF形式でダウンロード (3745K) -
Sachiko Okuno, Takako Kitani, Hitoshi Fujisawa1997 年 121 巻 1 号 p. 155-160
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe existence of isoforms of calmodulin-dependent protein kinase kinase (CaM-kinase kinase) in the rat brain was recently suggested by Northern and Western blot analyses and immunotitration [Okuno, S., Kitani, T., and Fujisawa, H. (1996) J. Biochem. 119, 1176 -1181] . In the present study, CaM-kinase kinase β, distinct from CaM-kinase kinase α which had been purified and cloned from rat cerebral cortex, was purified approximately 5, 000-fold from rat cerebellum and its properties were examined. The purified CaM-kinase kinase β gave a doublet at positions corresponding to molecular weights of 66, 000 to 67, 000 on SDSPAGE, and neither protein band reacted with antibody against CaM-kinase kinase α. Both CaM-kinase kinase α and β markedly activated both CaM-kinase I and IV, but CaM-kinase kinase β activated CaM-kinase IV more strongly than did CaM-kinase kinase α. The maximal extents of the activation of CaM-kinase I and IV by CaM-kinase kinase β were almost the same as those by CaM-kinase kinase α, suggesting that the two CaM-kinase kinases activated CaM-kinase I and IV by the same mechanisms.抄録全体を表示PDF形式でダウンロード (1924K) -
Shinya Oue, Akihiro Okamoto, Yumi Nakai, Masakiyo Nakahira, Takeji Shi ...1997 年 121 巻 1 号 p. 161-171
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe gene for aromatic amino acid aminotransferase (ArAT) from Paracoccus denitrificans was cloned, sequenced, and overexpressed in Escherichia coli cells. The sequence differed from that reported previously [Takagi, T., Taniguchi, T., Yamamoto, Y., and Shibatani, T. (1991) Biotechnol. Appl. Biochem. 13, 112-119]. The enzyme (pdArAT) was purified to homogeneity, and characterized. It was similar to aspartate aminotransferase (AspAT) and ArAT of E. coli (ecArAT) in many respects, including gross protein structure and spectro-scopic properties. pdArAT showed activities toward both dicarboxylic and aromatic substrates, and analysis of the binding of substrate analogs and quasisubstrates to the enzyme showed that both dicarboxylic and aromatic substrates take a similar orientation in the active site of pdArAT; these properties are essentially identical with those of ecArAT. As in the case of ecArAT, neutral amino acids with larger side chains are better substrates for pdArAT, suggesting that hydrophobic interaction between the substrate and the enzyme is important for the recognition of substrates with neutral side chains. pdArAT catalyzed transamination of phenylalanine and tyrosine far more efficiently (102-fold in terms of kcat/ Km) than those of straight-chain aliphatic amino acids with similar side-chain surface area, whereas ecArAT did not show significant preference for aromatic amino acids over aliphatic amino acids. This shows that the substrate-side-chain-binding pocket of pdArAT, as compared with the pocket of ecArAT, is well suited in shape for interaction with the phenyl and hydroxyphenyl rings of substrates. Thus, pdArAT is an ideal enzyme among ArATs for the study of the high-specificity recognition of two different kinds of substrates, the one having a carboxylic side chain and the other having an aromatic side chain.抄録全体を表示PDF形式でダウンロード (1490K) -
Mineko Tomomura, Akito Tomomura, Dewan Abdullah Abu Musa, Masahisa Hor ...1997 年 121 巻 1 号 p. 172-177
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーSystemic carnitine-deficient juvenile visceral steatosis (JVS) mice exhibit decreased expression of some liver-selective genes including those for the urea cycle enzymes during the infantile period. At 25 days, carbamoylphosphate synthetase (CPS) mRNA level was remarkably low in the liver of JVS mice, and the HNF-4 and C/EBP-α mRNA contents were also reduced. HNF-3α and C/EBP-R mRNAs were slightly higher in the liver of JVS mice, and HNF-1 mRNA remained normal. These results, together with the developmental changes of these transcription factor mRNA levels, suggest that HNF-4 and C/EBP-α are involved in the suppression of CPS expression. If JVS mice survived the crisis at 4-5 weeks, their body weight caught up with that of control mice around 7 weeks. The steady-state levels of CPS and argininosuccinate synthetase (ASS) mRNAs in the liver of JVS mice were normalized by no later than 8 weeks. Starvation for 48 h caused an increase of about twofold in CPS and ASS mRNA levels in the liver of control mice, while the same treatment failed to increase their levels in the liver of JVS mice. The starvation similarly caused increases in HNF-4 and C/EBP-α mRNA levels in the liver of both control and JVS mice, but the increases were significantly less in JVS mice than in control mice. Thus, the lack of induction of CPS and ASS mRNAs during development and under starvation in JVS mice correlated with the lower induction of HNF-4 and C/EBP-α mRNAs, and of HNF-4 and C/EBP-β mRNAs, respectively. Furthermore, all these changes seemed to correlate with the presence of fatty liver and the high serum free fatty acid levels, suggesting that disturbance of fatty acid metabolism affects nitrogen metabolism at least in part via altered gene expression of transcription factors such as HNF-4, C/EBP-α, and C/EBP-β.抄録全体を表示PDF形式でダウンロード (2788K)
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