巻号一覧
122 巻, 3 号
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Yoshiaki Ohkuma1997 年 122 巻 3 号 p. 481-489
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーGeneral transcription factors together with RNA polymerase II assemble on the promoter DNA and initiate transcription accurately in response to a variety of signals. Such signals enhance preinitiation complex formation by targeting components thereof via several alternative pathways. Two components of the initiation complexes, TFIIE and TFIIH, are known to function at both a late stage of transcription initiation and the following promoter clearance. TFIIH has been studied extensively because of its multiple enzymatic activities, functioning not only in transcription but also in nucleotide excision repair and cell cycle control. Fewer data have been reported for TFIIE, but its potential regulatory function as to TFIIH warrants further attention. In this review, an overall perspective of the functional roles of TFIIE and TFIIH during transcription initiation and the following promoter clearance will be presented as it has emerged from recent studies.抄録全体を表示PDF形式でダウンロード (4687K) -
Kimikazu Hashino, Hideyuki Matsushita, Ikunoshin Kato1997 年 122 巻 3 号 p. 490-493
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーTwo kinds of cells, human epidermoid carcinoma A431 cells and simian kidney COS-7 cells, were transfected with the chloramphenicol acetyl transferase (CAT) gene by electroporation, and then cultivated on culture dishes coated with two different forms of recombinant fibronectin fragments consisting of cell-binding domain (C-274) or heparin-binding domain and CS1 region (H-296). In the case of A431 cells, H-296-coated dishes significantly increased the amount of expressed CAT and the adhesion of electroporated cells in comparison with non-coated dishes. C-274 was effective for COS-7 cells. Overall, these fibronectin fragments increased the recovery of the transfectants on A-431 cells and COS-7 cells, respectively.抄録全体を表示PDF形式でダウンロード (475K) -
Masayuki Takeuchi, Hitoshi Fujisawa1997 年 122 巻 3 号 p. 494-497
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーαKAP is a protein produced from a gene within the gene of the α isoform of calmodulin-dependent protein kinase II (CaM-kinase IIα). It consists of the association domain of CaM-kinase IIα and a highly hydrophobic amino-terminal stretch consisting of 25 amino acids which is absent from CaM-kinase IIα. We previously demonstrated that αKAP is an integral membrane protein by subcellular fractionation analysis [Sugai, R., Takeuchi, M., Okuno, S., and Fujisawa, H. (1996) J. Biochem. 120, 773-779], but the exact subcellular localization of αKAP was not well understood. Here we demonstrate that αKAP is localized on the nuclear membrane of COS-7 cells transiently expressing αKAP. The nuclear membrane and perinuclear small vesicles were immunostained with an antibody against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids of αKAP. In contrast to the intact αKAP, the mutant αKAP, from which the hydrophobic aminoterminal segment had been deleted, accumulated within nuclei. Thus, αKAP may function as an anchoring protein for CaM-kinase II and/or other proteins in the perinuclear membrane.抄録全体を表示PDF形式でダウンロード (2400K) -
Takayuki Maruyama, Toshiya Kanaji, Shinji Nakade, Tomio Kanno, Katsuhi ...1997 年 122 巻 3 号 p. 498-505
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe effects of a novel membrane-penetrable modulator, 2APB (2-aminoethoxy diphenyl borate), on Ins (1, 4, 5) P3-induced Ca2+ release were examined. 2APB inhibited Ins (1, 4, 5)-P3-induced Ca2+ release from rat cerebellar microsomal preparations without affecting [3H]Ins (1, 4, 5) P3 binding to its receptor. The IC50, value (concentration producing 50% inhibition) of 2APB for inhibition of Ins (1, 4, 5) P3 (100nM) induced Ca2+ release was 42 μM. Further increase in the concentration of 2APB (more than 90μM) caused a gradual release of Ca2+ from cerebellar microsomal preparations. Addition of 2APB to the extracellular environment inhibited the cytosolic Ca2+ ([Ca2+]c) rise in intact cells such as human platelets and neutrophils stimulated by thromboxane-mimetic STA2 or thrombin, and leukotriene B4 (LTB4) or formyl-methionine-leucine-phenylalanine (FMLP), respectively. 2APB inhibited the contraction of thoracic aorta isolated from rabbits induced by angiotensin II (All), STA2, and norepinephrine in a non-competitive manner, but showed no effect on the contraction of potassium-depolarized muscle. 2APB had no effect on the Ca2+ release from the ryanodine-sensitive Ca2+ store prepared from rat leg skeletal muscle and heart. Although the specificity of 2APB with respect to the intracellular signaling system was not fully established, 2APB is the first candidate for a membrane-penetrable modulator of Ins (1, 4, 5) P3 receptor, and it should be a useful tool to investigate the physiological role of the Ins (1, 4, 5) P3 receptor in various cells.抄録全体を表示PDF形式でダウンロード (989K) -
Ken-ichi Yoshida, Shuichi Kawamura, Yoichi Mizukami, Masafumi Kitakaze ...1997 年 122 巻 3 号 p. 506-511
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーIschemic preconditioning is a phenomenon in which one or several cycle (s) of brief ischemia-reperfusion protects the myocardium against the cell injury caused by subsequent prolonged ischemia. Protein kinase C (PKC) inhibitors blunt the cardioprotection arising from ischemic preconditioning. To investigate which PKC isoform is involved in ischemic preconditioning, we identified the PKC isoform that translocates to the membrane fraction by means of immunoblotting with specific antibodies. PKC-α, δ, ε isoforms all increased in the membrane fraction after three cycles of 3min ischemia and 5min reperfusion (ischemic preconditioning) in the perfused rat heart. The ischemic preconditioning significantly improved the recovery of left ventricular developed pressure (LVDP) during reperfusion following 20min of ischemia. A PKC specific inhibitor, chelerythrine (1.0μM) blocked the effect of ischemic preconditioning on LVDP recovery and the translocation of PKC- α, δ, ε isoforms. These data suggest that one or more of these three isoforms of PKC is involved in ischemic preconditioning by phosphorylating membrane proteins.抄録全体を表示PDF形式でダウンロード (1641K) -
Tadahiro Ohmura, Tadashi Ueda, Hiroyuki Motoshima, Tomohiro Tamura, Ta ...1997 年 122 巻 3 号 p. 512-517
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーHis 15 of hen lysozyme is located at the protein surface and is partly buried by the neighboring residues. The side chain of His 15 forms hydrogen bonds with surrounding residues and these hydrogen bonds are somewhat buried. A series of mutant lysozymes at the position 15 (Gly, Ala, Val, and Phe) was prepared, and their stabilities were analyzed by GdnHCI denaturation and X-ray crystallography. The mutants were less stable than the wild type at pH 5.5 and 35°C. In H15G and H15A, X-ray crystallography revealed two fixed water molecules at the mutated region, which formed similar hydrogen bonds to those in the wild type. On the other hand, it was suggested that the hydrogen bonds were disrupted and that several unfavorable van der Waals' contacts occurred in H15V and H15F. Therefore, we concluded that His 15 stabilized the lysozyme structure by forming hydrogen bonds and the best packing with the neighboring residues. Moreover, we found that the method of protein stabilization by increasing the hydrophobicity of an amino acid residue was not always effectively applicable, especially when the residue had formed a hydrogen bond.抄録全体を表示PDF形式でダウンロード (651K) -
Hirotaka Fujimoto, Issei Mabuchi1997 年 122 巻 3 号 p. 518-524
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーWe have developed a method for the isolation of cleavage furrows from dividing sea urchin eggs, which is applicable to various sea urchin species. The new method differs from that used for isolating cleavage furrows from sand dollar Clypeaster japonicus eggs [Yonemura, S., Mabuchi, I., and Tsukita, S. (1991) J. Cell Sci. 100, 73-84] in the type and concentration of detergent included in the isolation medium, the temperature during the treatment of dividing eggs with the isolation medium, and the centrifugation conditions. The contractile ring was included in the isolated cleavage furrows, as seen on rhodaminephalloidin staining of actin filaments. When the furrows were isolated with the isolation medium containing both NaF and β-glycerophosphate, which are potent protein phosphatase inhibitors, the isolated furrows were found to be accompanied by the mitotic apparatus. When the isolation was carried out in the absence of both NaF and β-glycerophosphate, cleavage furrows without the mitotic apparatus were obtained. The development of a method of isolation of cleavage furrows from regular sea urchin eggs enabled us to compare protein constituents among furrows from different sea urchin and sand dollar species. We found that 32, 36, and 51 kDa proteins were concentrated in common in the cleavage furrows isolated from eggs of the sand dollars, C. japonicus and Scaphechinus mirabilis, and the sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus nudus, on two-dimensional gel electrophoreses.抄録全体を表示PDF形式でダウンロード (3832K) -
Hye-Won Shin, Chisa Shinotsuka, Seiji Torii, Kazuo Murakami, Kazuhisa ...1997 年 122 巻 3 号 p. 525-530
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe dynamin family of GTP-binding proteins are implicated in vesicular transport. These include mammalian dynamins I, II, III, and yeast Vps1p and Dnm1p. Dynamin is involved in the formation of clathrin-coated vesicles at the plasma membrane. On the other hand, Vps1p and Dnm1p appear to be involved in transport from the late Golgi compartment to vacuoles and in an endocytic process, respectively. In this study, we identified a novel human protein, named Dnm1p/Vps1p-like protein (DVLP). It resembled more closely Dnmlp and Vpslp than dynamins not only in the primary structure but also in the domain organization. DVLP mRNA was expressed ubiquitously, suggesting that this protein plays a fundamental role in cellular function. Immunofluorescence analysis of cells expressing epitope-tagged DVLP revealed that it showed a diffused perinuclear staining pattern that was not superimposed on that of the marker protein for the Golgi apparatus, trans-Golgi network, lysosomes, endosomes, or endoplasmic reticulum. These data suggest that DVLP is not involved in the formation of known coated vesicles.抄録全体を表示PDF形式でダウンロード (4174K) -
Takeshi Tomita, Shingo Tsuyama, Yoshio Imai, Teizo Kitagawa1997 年 122 巻 3 号 p. 531-536
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーSoluble guanylate cyclase (sGC) consisting of two different subunits (α: Mr=74, 000, β: Mr=69, 000) was purified more than 12, 000-fold in terms of specific activity from the supernatant of bovine lung homogenates and characterized. The heme content determined with the pyridine hemochromogen method and Bradford's protein assay was 0.8 heme per dimer. Cholera, pertussis, and botulinum C3 toxins modified exclusively the β-subunit of sGC, yielding the ADP-ribose-bound compound with 1:1 stoichiometry, and Vmax for the cyclase reaction was increased 10 times by this modification. When the ADP-ribosylation of sGC was performed simultaneously with two or three bacterial toxins which have distinct amino acid specificities, the resultant enzyme had only one ADP-ribose, and the activity was the same as that of the enzyme modified with one toxin. When NO was incorporated into the reaction mixture containing the ADP-ribosylated sGC, the cyclase activity noticeably increased by approximately the same amount as that seen for the unmodified enzyme. Such effects were not seen with CO. When ADP-ribosylated sGC was incubated with Mn2+, the enzyme activity was synergistically increased. The heme-deleted sGC was also ADP-ribo-sylated by bacterial toxins and its activity was raised. These findings suggest that sGC has an ADP-ribosylation site near the GTP binding site, like other GTP-binding proteins, and that the β-subunit regulates the activity.抄録全体を表示PDF形式でダウンロード (2930K) -
Takamitsu Yorifuji, Tohru Ishihara, Takashi Naka, Shinya Kondo, Eiichi ...1997 年 122 巻 3 号 p. 537-543
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーPolyamine aminotransferase of Arthrobacter sp. TMP-1 was induced by 1, 3-diaminopropane (DAP), N-3-aminopropyl-1, 3-diaminopropane (norspermidine), spermidine, and spermine, but not by putrescine. The enzyme was purified to homogeneity. Its molecular weight and subunit size were 129, 000 and 64, 000, respectively. Its absorption spectrum had maxima at 280 and 420nm and a shoulder at about 350nm, and changes were observed upon the addition of DAP, putrescine, and sodium borohydride. The spectrum and its changes indicated that the enzyme contained pyridoxal-5'-phosphate as the coenzyme. The coenzyme content was found to be 1 mol per mol of subunit. DAP, putrescine, norspermidine, spermidine, and spermine were active amino donors and gave relative rates of 100, 73, 24, 30, and 23%, respectively. Pyruvate was the most active amino acceptor, while 2-ketoglutarate and oxaloacetate were inert. The equilibrium constant of the DAP-pyruvate transamination was 0.34. DAP was suggested to be a minor product of the norspermidinepyruvate reaction.抄録全体を表示PDF形式でダウンロード (1000K) -
Takamitsu Yorifuji, Shinya Kondo, Takashi Naka, Tohru Ishihara, Eiichi ...1997 年 122 巻 3 号 p. 544-549
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe norspermidine-pyruvate reaction catalyzed by polyamine aminotransferase from Arthrobacter sp. TMP-1 formed N-3-aminopropyl-3-aminopropionaldehyde (APAPAL), L-alanine, 1, 3-diaminopropane (DAP), allylamine, and acrolein, and the relative rates of formation of the latter four products were 24, 3.3, 2.3, and 1.2%, respectively, of the rate of the DAP-pyruvate transamination. The identification of APAPAL was done by 13C-NMR after it had been enzymatically oxidized to N-3-aminopropyl-β-alanine followed by isolation of the oxidized product. The DAP was also isolated and identified by 13C-NMR. The allylamine and acrolein were identified by HPLC and a specific color reaction with m-aminophenol, respectively. In the absence of pyruvate, the enzyme catalyzed the elimination of DAP from norspermidine to yield allylamine, and the addition of DAP to allylamine to yield norspermidine with relative rates of 0.007 and 0.095%, respectively. When allylamine was incubated with the enzyme as the sole substrate, it was converted to N-allyl-1, 3-diaminopropane and an unidentified product.抄録全体を表示PDF形式でダウンロード (2233K) -
Akemi Nasuda-Kouyama, Munehiro Nakata, Tetsutaro Iizuka, Yasuhiro Isog ...1997 年 122 巻 3 号 p. 550-555
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーTo investigate the mechanism of nitroso compound-induced inhibition of the respiratory burst in neutrophils, we studied fluorescence quenching of perylene by nitroso-compounds in the membrane fractions of neutrophils at 17, 27, and 37°C and the reagent-induced inhibition of superoxide generation at 28 and 37°C. With increasing temperature, the quenching of perylene fluorescence and inhibition of superoxide generation by nitrosobenzene (NB) were both diminished, while those by 2-nitrosotoluene (NT) were both enhanced. The temperature dependence of the inhibition constants and the quenching constants indicates that the binding of NB is exothermic (ΔH=-27 kJ/mol for inhibition and ΔH=-29 kJ/mol for quenching) and essentially enthalpy-driven. On the other hand, that of NT is endothermic (ΔH=+16 kJ/mol for inhibition and quenching) and essentially entropydriven. Quenching studies of perylene fluorescence in synthetic vesicles made of endogenous polar lipids of neutrophils showed that the enthalpy changes of NB- and NT-binding with perylene in lipids were similar to each other. Moreover, their values were in good agreement with that of NT, but not of NB, in the membrane fractions, an assembly of proteins and lipids, of neutrophils. These results suggest that NB inhibits the activity by binding to proteins in the membrane, whereas inhibition by NT occurs through hydrophobic interaction with lipids and/or proteins.抄録全体を表示PDF形式でダウンロード (698K) -
Taiichi Sakamoto, Gota Kawai, Masato Katahira, Mi Hee Kim, Yoichiro Ta ...1997 年 122 巻 3 号 p. 556-562
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーAn RNA 28-mer (Rz28) was obtained as a major product by in vitro transcription with T7 RNA polymerase of a promoter-template DNA, which contains a sequence for the enzyme component, RNA 24-mer (Rz24), of a mutant hammerhead ribozyme system. Sequence analysis and enzymatic probing study showed that Rz28 has 4 extra nucleotides at the 3'-terminus, the sequence of which is complementary to that of the 5'-terminal sequence of Rz24, and forms a stable hairpin structure. NMR studies using a 15N-guanine-labeled derivative suggested that Rz28 contains tandem G:A pairs that are not of the side-by-side type which is found in the crystal structure of hammerhead ribozyme complexes. Comparison of the HMQC spectra of 15N-guanine-labeled Rz28 and Rz24 suggested that Rz24 also contains the same type of tandem G:A pairs.抄録全体を表示PDF形式でダウンロード (2172K) -
Gregory J. Wilson, Sarah E. Shull, Nariman I. Naber, Roger Cooker1997 年 122 巻 3 号 p. 563-571
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーInteractions between the two myosin heads were studied in skinned rabbit slow-twitch muscle fibers activated in the presence of vanadate (Vi), a phosphate analog. The strong complex between Vi, MgADP, and myosin trapped the myosin in an inactivated myosinMgADP•Vi state. Electron paramagnetic resonance spectroscopy was used to quantitate the fraction of myosin heads trapped in the presence of a spin labeled analog of ATP (SLATP). Force was found to depend directly on the fraction of untrapped heads. At high [Vi] (low force), most untrapped heads would have a trapped partner. The equivalence of force with the proportion of untrapped heads shows that the isometric force produced by a single untrapped myosin head on a molecule with a trapped partner is equivalent to that produced by either head of a myosin molecule with neither head trapped. The actin-activated MgATPase activities of one-headed and two-headed skeletal myosin species were inhibited similarly by Vi, suggesting that trapping one head did not preclude trapping its partner. These data indicate that the two skeletal muscle myosin heads can function without interacting during maximal Ca2+-activated force generation.抄録全体を表示PDF形式でダウンロード (1336K) -
Masateru Nishihara, Yosuke Koga1997 年 122 巻 3 号 p. 572-576
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe enzyme which seems to be responsible for the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids was purified from a methanogenic archaeon, Methanobacterium thermoautotrophicum, and characterized. The enzyme, sn-glycerol-1-phosphate: NAD (P)+ oxidoreductase (sn-glycerol-1-phosphate dehydrogenase), was purified 7, 600-fold from a cell free extract by ammonium sulfate fractionation and seven steps of chromatography. The final preparation exhibited a specific activity of 617 μsmol/min/mg (Vmax) and gave a single band corresponding to 38 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme showed an apparent molecular mass of 302 kDa on gel-filtration chromatography, indicating it is present as a homooctamer. Maximum activity was observed at 75°C at near neutral pH. The activity was stimulated by potassium ions. The Km for dihydroxyacetone phosphate was 7.5 times smaller than that for sn-glycerol-1-phosphate, suggesting that the formation of sn-glycerol-1-phosphate is the natural direction in the cell. Under the assay conditions used, no product inhibition was observed. The N-terminal amino acid sequence was determined.抄録全体を表示PDF形式でダウンロード (617K) -
Naotaka Hamasaki, Kenshi Okubo, Hiroyuki Kuma, Dongchon Kang, Yoshiaki ...1997 年 122 巻 3 号 p. 577-585
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーTo assess the fidelity of hydropathy prediction for band 3 protein, we determined the cleavage sites of the protein and the portions of the protein tightly bound to the membrane lipid bilayer by means of in situ proteolytic digestion. For the removal of all anticipated hydrophilic connector loops from membranes, we had to denature the band 3 protein molecule in situ by alkali treatment. When the alkali-treated membranes were digested with trypsin, chymotrypsin, and pepsin, the majority of the anticipated transmembrane portions remained in the membrane fraction. However, five anticipated transmembrane portions were released into the supernatant fraction. Thus, the first, second, third, sixth and tenth anticipated transmembrane portions, in accordance with the hydropathy prediction, were released into the supernatant with the proteolytic digestion method. This indicates that these anticipated transmembrane portions are not bound with the boundary lipids although the hydrophobicity of these portions is comparable to that of the portions experimentally remaining in the membrane fraction. It is conceivable that the membrane peptide portions of band 3 protein could be classified into at least two categories, i. e. one bound to the boundary lipids and the other free from the boundary lipids. Approximately 90% of the transmembrane domain of the band 3 protein are recovered in either the supernatant fraction or the membrane fraction. The fidelity of hydropathy prediction for polytopic membrane proteins and the nature of the membrane embedded peptide portions are discussed.抄録全体を表示PDF形式でダウンロード (977K) -
Akiko Yamamoto, Yumi Ando, Ken-ichi Yoshioka, Kouhei Saito, Takuya Tan ...1997 年 122 巻 3 号 p. 586-594
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーHigh mobility group (HMG) proteins 1 and 2 contain two similar but non-identical repeats of DNA-binding domains and an acidic C-terminal. The proposed functions of HMG proteins 1 and 2 imply a probable difference in their DNA-binding abilities. The primary studies by gel retardation assay showed that HMG2 has higher affinity than HMG1 for supercoiled and linear DNA. The DNA-binding of HMG2 appeared strong enough to allow exchange with HMG1 molecule already bound to DNA, while the DNA-binding region of HMG1 showed higher affinity than that of HMG2. In order to compare more quantitatively the affinities, surface plasmon resonance (SPR) measurements using a BlAcore instrument were conducted. The kinetic data indicated that the Kd for the complex of HMG2 with DNA is smaller than that of HMG1, in contrast to the situation for the DNA-binding region of these proteins. The sequence between the second DNA-binding domain and the acidic C-terminal of HMG proteins is required for tight DNA-binding. Also, the acidic C-terminal strongly modulates the DNA-binding ability of each protein. The usefulness of SPR measurement for quantitative analysis of affinity and regions involved in DNA-binding under conditions nearly identical to those in solution is discussed.抄録全体を表示PDF形式でダウンロード (4064K) -
Toshimasa Tsujinaka, Masanori Kishibuchi, Masahiko Yano, Takashi Morim ...1997 年 122 巻 3 号 p. 595-600
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーSerum IL-6 level increased after the injection of turpentine oil into the right gastrocnemius muscle in mice. The mRNA level of IL-6 was highest in the injected muscle at 12h after injection, but was not identified in the opposite muscle. The activities of cathepsins B and B+L started to elevate after 12h in the injected muscle and markedly increased after day 3. Likewise, the mRNA levels of cathepsins B and L markedly increased from day 1 to day 5 in the injected muscle. However, a very mild increase was also observed in the opposite muscle. Immunohistochemical staining of cathepsins B and L exhibited positive reactions as fine granules in myofibers at 12h and strong positive reactions in the infiltrating macrophages at 3 days. Atrophy of myofibers type 1 and 2 was evident in a time-dependent manner in the injected muscle. Treatment with rat anti-mouse IL-6 receptor monoclonal antibody inhibited the increase in cathepsin activities in the injected muscle. We conclude that IL-6 produced in the inflamed muscle is involved in the process of muscle degeneration, especially through the activation of lysosomal cathepsins.抄録全体を表示PDF形式でダウンロード (2934K) -
Tsutomu Kabashima, Ana Kitazono, Atsuko Kitano, Kiyoshi Ito, Tadashi Y ...1997 年 122 巻 3 号 p. 601-605
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーWe cloned and sequenced the Serratia marcescens prolyl aminopeptidase (SPAP) gene. Nucleotide sequence analysis revealed an open reading frame of 951 bp, encoding a protein of 317 amino acids with a predicted molecular weight of 36, 083. The expressed enzyme was purified about 90-fold on columns of Toyopearl HW65C and DEAE-Toyopearl, with an activity recovery of 30%. The apparent molecular weight of the purified enzyme was 36, 000 and 38, 000 as estimated by SDS-PAGE and gel filtration, respectively. The enzyme was not inhibited by diisopropyl phosphofluoridate (DFP) or phenylmethylsulfonyl fluoride (PMSF), but was markedly inhibited by 3, 4-dichloroisocoumarin (DCIC). Crystals of the enzyme were grown by the hanging drop vapor diffusion method using PEG6000 as a precipitant at pH 6.5. The crystals are tetragonal with cell dimensions a=b=65.6 Å, and c=169.81 Å, a space group P41212 or P43212, and probably contain one monomer in the asymmetric unit. They diffract to at least 2.22 Å resolution.抄録全体を表示PDF形式でダウンロード (1785K) -
Kazuko Hori, Toshitsugu Kurotsu1997 年 122 巻 3 号 p. 606-615
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーAn aggregation substance of gramicidin S synthetases was found and purified by DEAE-cellulose chromatography and CM-chromatography from cell debris of Bacillus brevis Nagano. It specifically aggregated and inactivated gramicidin S synthetases 1 (GS1) and 2 (GS2). On the basis of amino acid composition analysis, reversed-phase HPLC, FAB mass spectrometry, amino acid sequence analysis, and antibacterial activity, this substance (GrS-aggregation substance) was identified as gramicidin S. A gramicidin S derivative bearing a lysine residue in place of one ornithine residue was also detected as a minor component of GrS-aggregation substance. The extent of the aggregation was dependent on the concentration and relative amount of gramicidin S. The inhibition of the enzyme activities was irreversible and the inhibition was proportional to the amount of gramicidin S, like the aggregation of the enzymes. The degree of GS2 inhibition in the amino aciddependent ATP-PPi exchange reaction varied with the amino acids of gramicidin S and increased in order of the amino acid sequence of gramicidin S. The degree of inhibition of the overall synthesis of gramicidin S was the same as that in the leucine-dependent exchange reaction.抄録全体を表示PDF形式でダウンロード (6521K) -
Naoki Kanayama, Yasukazu Himeda, Haruyuki Atomi, Mitsuyoshi Ueda, Atsu ...1997 年 122 巻 3 号 p. 616-621
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーIn the n-alkane-assimilating yeast Candida tropicalis, there are two isozymes of acetoacetyl-CoA thiolase, peroxisomal acetoacetyl-CoA thiolase (peroxisomal Thiolase I), and cytosolic acetoacetyl-CoA thiolase (cytosolic Thiolase I). We have previously isolated two genes (CT-T1A and CT-T1B) which encode Thiolase I. In order to compare the expressed products of Thiolase I isozyme-encoding genes in C. tropicalis, cytosolic Thiolase I was first purified from glucose-grown C. tropicalis in which the proliferation of peroxisomes and the expression of peroxisomal Thiolase I were repressed. Cytosolic Thiolase I was virtually identical to peroxisomal Thiolase I in molecular mass, kinetic and immuno-chemical properties, and primary structure at the N-terminus. Amino acid sequence analysis revealed that cytosolic Thiolase I was the mixture of products of two genes (CT-T1A and CT-T1B), as in the case of the peroxisomal enzyme. CT-T1A and CT-T1B were expressed independently in the yeast Saccharomyces cerevisiae and the recombinant proteins were purified. Recombinant Thiolase IA and IB exhibited practically identical enzymatic properties to cytosolic and peroxisomal Thiolase Is from C. tropicalis. These results revealed that cytosolic Thiolase I and peroxisomal Thiolase I were encoded not by different genes, but by the same genes (CT-T1A and CT-T1B) and are present as a mixture of products expressed by both genes, although their subcellular localizations are different.抄録全体を表示PDF形式でダウンロード (3022K) -
Midori Yokoyama-Kobayashi, Mihoro Saeki, Shingo Sekine, Seishi Kato1997 年 122 巻 3 号 p. 622-626
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーRecently, we developed a simple method for detecting a secretory signal sequence encoded by a cDNA fragment. In this study, we used this method to select cDNA clones encoding secretory proteins from a human full-length cDNA library. Full-sequencing analysis of the candidate clones revealed that one clone encoded a novel TGF-β superfamily protein. The clone encodes a protein of 308 amino acids of which the C-terminal region shows a characteristic feature of TGF-β superfamily proteins: seven conserved cysteine residues at the C-terminal preceded by a putative processing site composed of a basic amino acid repeat. The corresponding transcripts are highly expressed in the placenta, so the novel protein may play an important role in reproduction.抄録全体を表示PDF形式でダウンロード (3345K) -
Yukiyasu Asano, Akira Ishihama1997 年 122 巻 3 号 p. 627-634
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーInfluenza virus RNA polymerase is a multifunctional and multisubunit enzyme consisting of three viral P proteins, PB1, PB2, and PA. We have previously shown that radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB1 subunit. Here we confirmed the specific crosslinking of PB1 using oxidized GTP and further identified the GTP analogue-binding domains after proteolytic cleavage of the crosslinked PB1 with V8 protease. The cleavage pattern of PB1 was determined by analysis of the amino-terminal proximal sequence of fragments generated in the presence of increasing concentrations of V8 protease. The GTP-crosslinking was identified in three fragments: two adjacent fragments, P6 starting from residue 179 and P11b starting from residue 298; and the third fragment, P11c, starting from residue 458. Thus, we propose that two GTP-binding sites exist in the PB1 subunit, i. e., the amino terminal-proximal site I located at the boundary between P6 and P11b, and the carboxy terminal-proximal site II on P11c fragment. The locations of GTP-binding sites I and II are close to those of sequence motif A and motif D, respectively, conserved among RNA-dependent RNA polymerases. Of the two fragments forming site I, the crosslinking of 8-N3 GTP is higher to P11b, while that of oxidized GTP is higher to P6, suggesting that the ribose and guanine moieties of GTP bound in this binding pocket face P6 and P11c, respectively. From the V8 concentration-dependent change in proteolytic cleavage pattern, it is likely that the two GTP-binding sites on PB1 protein are located on structurally different domains. The existence of two GTP-binding sites is discussed in relation to the binding sites for substrates, primers, and products.抄録全体を表示PDF形式でダウンロード (4903K) -
Tomoko Sonoda, Kazuko Kita, Sumio Ishijima, Toshiharu Ishizuka, Imtiaz ...1997 年 122 巻 3 号 p. 635-640
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーRat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of two catalytic subunits (PRS I and II, in a ratio of approximately 4:1) and two catalytically inactive PRPP synthetase-associated proteins. To better understand the significance of the complex structure, the properties of the native liver enzyme were compared with those of homologous aggregates of recombinant PRS I and PRS II (rPRS I and rPRS II). (1) The specific activity per catalytic subunits of the liver enzyme was about 2.5 times lower than that of rPRS I over a wide pH range. Km values for substrates and Ka values for Pi and Mg2+ of the three enzymes were similar. (2) Specific activity of the liver enzyme for the reverse reaction was about 2 times lower than those of rPRSs. Km values for substrates of the three enzymes were comparable. (3) The liver enzyme was more stable than were rPRSs when incubated at a high temperature or in the absence of stabilizing agents. (4) The liver enzyme was markedly less sensitive to inhibition by nucleotides compared to rPRS I. GDP at 1mM inhibited the liver enzyme and rPRS I by 32 and 93%, respectively. This effect is not ascribable to molecular interaction between rPRS I and II, as reconstitution of the two did not alter the sensitivity to nucleotide inhibition. (5) Our observations suggest that complex aggregation states of the native enzyme not only suppress the activities but also stabilize the catalytic subunits and the associated proteins and remarkably reduce the sensitivity to inhibition by nucleotides.抄録全体を表示PDF形式でダウンロード (836K) -
Shigemi Yoshioka, Taizo Suzuki, Masao Kawakita1997 年 122 巻 3 号 p. 641-646
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーDistribution of the NHE1 and the NHE3 isoforms of Na+/H+ exchanger in the plasma membranes of bovine kidney cortex was analyzed. Fractionation of the plasma membranes by centrifugation on a Percoll density gradient resulted in clear separation of the basolateral membranes (BLM) from the brush-border membranes (BBM), with Na+, K+-ATPase and aminopeptidase M as their respective marker enzymes. Under these conditions, a 110 kDa protein cross-reactive with an anti-NHE1 antibody was detected exclusively in the BLM fractions, while a 90 kDa protein cross-reactive with an anti-NHE3 antibody was detected in the BBM fractions. A conventional Mg2+-precipitation method for obtaining the BBM, which is adequate with rabbit kidney as a starting material, turned out to be inadequate with bovine kidney cortex, since a considerable amount of the 110-kDa NHE1 protein was detected in the bovine kidney BBM fraction prepared by this procedure, together with the 90-kDa NHE3 protein. Percoll density gradient centrifugation is thus strongly recommended for the fractionation of BBM and BLM of bovine kidney cortex. The bovine NHE1 isoform was shown to be unique in that it is far less sensitive to the inhibition by ethylisopropylamiloride than that of other species.抄録全体を表示PDF形式でダウンロード (3455K) -
Akihiro Michihara, Makoto Sawamura, Yasuo Nara, Katsumi Ikeda, Yukio Y ...1997 年 122 巻 3 号 p. 647-654
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe biosynthesis of cholesterol is regulated mainly by HMG-CoA reductase, however, recent studies indicated the pivotal role of another enzyme in cholesterol homeostasis. A previous report showed a marked decrease in the activity of mevalonate pyrophosphate decarboxylase (MPD) in stroke-prone spontaneously hypertensive rats and its possible involvement in the pathogenesis of the disorder. In this study, we purified liver MPD from rats fed a diet containing cholestyramine and pravastatin (CP diet) using conventional chromatographic techniques. We obtained two electrophoretically homogeneous enzyme preparations; 45 and 37kDa proteins with specific activities of 8.0 and 7.4μmol/min/mg, respectively. The enzymes showed similar molecular weights of 90 kDa, as judged on gel permeation chromatography. A kinetic study indicated apparent Km values for mevalonate pyrophosphate and ATP of 22.7μM and 0.71mM, respectively, for the 45 kDa MPD, and 20.0μM and 0.80mM, respectively, for the 37 kDa MPD. Half maximum activities were observed at 1.5mM and 1.1mM Mg2+ for the 45 and 37 kDa MPDs, respectively. Both enzymes required ATP as a phosphate acceptor, and in addition Mg2+, Mn2+, and Co2+ were effective as divalent cations. The optimum pH for both enzymes was 7.0. The isoelectric points for the 45 and 37 kDa MPDs were 5.6 and 5.4, respectively. Polyclonal antiserum raised against the 45 kDa enzyme detected both the 45 and 37 kDa bands on immunoblots with CP diet-induced liver crude extract as an antigen. However, non-induced liver contained the 45kDa protein but not the 37 kDa protein. These results indicated that the CP diet induced a new species, 37 kDa, of MPD which is characteristically and immunologically very similar to the well-known 45 kDa MPD.抄録全体を表示PDF形式でダウンロード (4787K) -
Mizuho Une, Masaki Konishi, Yasuyuki Suzuki, Shinjiro Akaboshi, Michik ...1997 年 122 巻 3 号 p. 655-658
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーBile acid profiles in serum, urine and bile from an infant with a peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-bifunctional protein) deficiency were analyzed by means of gas-liquid chromatography, gas-liquid chromatography-mass spectrometry, and high-performance liquid chromatography. As in such several peroxisomal disorders as Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, the accumulation of C27-bile acid intermediates was also demonstrated in the infant with D-bifunctional protein deficiency, accounting for 74% of the total bile acids in serum, 59% in urine, and 35% in bile. In addition, the major constituents of the C27-bile acids were (24R, 25R)- and (24R, 25S)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoic acids along with small amounts of their 24S counterparts. Since immunoreactive acyl-CoA oxidase, L-bifunctional protein, and thiolase were all present in the liver, the impairment of the oxidative side-chain cleavage in bile acid biosynthesis is considered to be due to the defect of D-bifunctional protein.抄録全体を表示PDF形式でダウンロード (521K) -
Hiroaki Eguchi, Shunji Kaya, Akira Shimada, Yuka Ootomo, Ken Nomoto, M ...1997 年 122 巻 3 号 p. 659-665
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーH+, K+-ATPase preparations from pig stomach were modified with a sulfhydryl fluorescence reagent, N-[p-(2-benzimidazolyl) phenyl] maleimide (BIPM). The addition of ATP to the modified enzyme preparations in the presence of Mg2+ decreased the BIPM fluorescence but increased the Trp fluorescence. After exhaustion of ATP, the fluorescence intensities increased and decreased to the original levels, respectively. The results of stopped flow and rapid quenching experiments suggested that the decrease in BIPM fluorescence (36/s) was accompanied by binding of Mg2+ and ATP or phosphorylation (35-36/s) which was followed by slower increases in Trp fluorescence (24/s) and light scattering (20/s). Tosylphenylalanyl chloromethyl ketone-trypsin treatment of the modified preparations, which showed an about 1% decrease in BIPM fluorescence accompanying phosphorylation, gave one major fluorescent peptide peak on reverse-phase chromatography. Amino acid sequence analysis of the peptide revealed the following sequence, Ser-Pro-Glu-X-Thr-His-Glu-Ser-Pro-Leu-Glu-Thr-Arg. On comparison with the amino acid sequence deduced from cDNA from pig stomach [Maeda, M., Ishizaki, J., and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157, 203-209], X was shown to correspond to Cys241 of the α-chain in H+, K+-ATPase. These data and others suggest that the decrease in BIPM fluorescence at Cys241 reflects some molecular event triggered by the binding of ATP with Mg2+ and/or phosphorylation, whereas the increases in the intrinsic Trp fluorescence and light scattering reflect one after phosphorylation.抄録全体を表示PDF形式でダウンロード (951K) -
Makoto Ushimaru, Yasuo Shinohara, Yoshihiro Fukushima1997 年 122 巻 3 号 p. 666-674
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーThe position in the acyl phosphate linkage of the phosphorylated intermediate of (Na+, K+)-ATPase that is cleaved by N-methylhydroxylamine was compared with that of the model compound acetylphosphate. The products of the cleavage of the phosphoenzyme by methylhydroxylamine were the active enzyme and a N-P compound, not the inhibited enzyme and inorganic phosphate. This means that the bond cleaved by methylhydroxylamine was the O-P bond, not the C-O bond. In contrast, methylhydroxylamine did not cleave the O-P bond of acetylphosphate in solution, at pH values from 0.3 to 7.0, whether or not the phosphoryl group formed a complex with magnesium. Acetylphosphate and hydroxylamine formed acetohydroxamic acid. Therefore, the state of the acyl phosphate bond in the native phosphoenzyme and in acetylphosphate in solution was different, and the difference was not due to different dissociation states of their phosphoryl groups or the binding of magnesium to the phosphoenzyme. Molecular orbital calculations for acetylphosphate revealed that the phosphorus atom charge is more positive than the carbon atom, irrespective of the dissociation state of the phosphoryl group. Similarly, the overlapping electron population of the O-P bond is always smaller than that of the C-O bond. Thus, the electronic structure of the acyl phosphate linkage of acetylphosphate under vacuum supports the results obtained with the native phosphoenzyme, rather than those obtained with acetylphosphate in solution. The linkage in the active site of the phosphorylated intermediate of (Na+, K+)-ATPase appeared to be equivalent to the non-hydrated state of the model compound acetylphosphate. The phosphoenzyme with bound ouabain, or without a tightly bound divalent cation was insensitive to methylhydroxylamine. The native phosphoenzyme of (Ca2+)-ATPase was not susceptible to methylhydroxylamine.抄録全体を表示PDF形式でダウンロード (2250K) -
Shigeki Higashiyama, Michiharu Horikawa, Kouji Yamada, Naohiro Ichino, ...1997 年 122 巻 3 号 p. 675-680
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーA novel member of the epidermal growth factor (EGF) family, the neural- and thymusderived activator for ErbB kinases (NTAK), has been purified and cloned. Five alternative spliced isoforms have been detected in the rat adrenal pheochromocytoma cell line, PC-12 cells. The rat NTAKα2a isoform exhibits 94% identity in its primary sequence with the human NTAKα isoform. In vivo, NTAK is only expressed in the brain of rat E11.5 embryos, and in the brain and thymus of adult rats. The soluble 46 kDa form binds directly to ErbB3 and B4, but not to ErbB1 or B2. NTAK, however, transactivates ErbB1 and B2 via heterodimerization with ErbB3 or B4. NTAK stimulates the differentiation of MDA-MB-453 cells and competitively inhibits the binding of [125I] neuregulin to these cells. In addition to these neuregulin-like properties, NTAK exhibits limited structural homology to neuregulins in the immunoglobulin (Ig)-like, EGF-like, and hydrophobic domains. Thus, NTAK appears to be a new member of the EGF family displaying neuregulin properties.抄録全体を表示PDF形式でダウンロード (4186K) -
1997 年 122 巻 3 号 p. 681
発行日: 1997年
公開日: 2008/11/18
ジャーナル フリーPDF形式でダウンロード (18K)
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