巻号一覧
118 巻, 4 号
選択された号の論文の29件中1～29を表示しています
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Makoto Komiyama1995 年 118 巻 4 号 p. 665-670
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーTotally synthetic and sequence-specific nucleases and ribonucleases, which hydrolyze DNAs and RNAs selectively at target sites, have been prepared. Lanthanide ions, which efficiently hydrolyze the phosphodiester linkages in nucleic acids, are attached to the 5'-end of synthetic DNA oligomers (sequence-recognizing sites) by use of an iminodiacetate ligand. Under physiological conditions, the hybrids selectively hydrolyze substrate DNA or RNA at the 3'-side of the sequence which is complementary with the DNA oligomers in the hybrids. The cerium (IV) ion is the most active as to DNA scission, whereas the europium (III), thulium (III), and lutetium (III) ions are the most effective for RNA scission. All the scissions proceed totally via hydrolysis of the phosphodiester linkages, in the same way as the scissions by natural nucleases and ribonucleases do. The artificial enzymes, which show far greater sequence-specificities than natural ones, exhibit high potential for application to molecular biology, biotechnology, and therapy.抄録全体を表示PDF形式でダウンロード (2857K) -
Katsuyuki Tanizawa1995 年 118 巻 4 号 p. 671-678
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーRecently, two novel quinonoid coenzymes, 2, 4, 5-trihydroxyphenylalanine quinone (topa quinone; TPQ) and tryptophan tryptophylquinone (TTQ), were identified in copper-containing amine oxidase and methylamine dehydrogenase, respectively. Unlike the formerly known quinonoid coenzyme, pyrroloquinoline quinone (PQQ), which is non-covalently bound to several prokaryotic dehydrogenases and produced through its own biosynthetic pathway, each of TPQ and TTQ is bound covalently to the polypeptide chain as an integral amino acid residue and encoded by a codon for a normal (unmodified) amino acid in the gene. Thus, these coenzymes must be generated through post-translational modification of the precursor amino acid; for TPQ, oxidation of a specific tyrosine occurring in the consensus Asn-Tyr-Asp/Glu sequence, and for TTQ, oxidation of a specific tryptophan and cross-linking with another tryptophan separated by 50 residues in the same polypeptide chain. We recently demonstrated that, using the inactive precursor forms of bacterial copper amine oxidases, TPQ is generated through self-processing of the protein with the participation of the bound copper ions. On the other hand, the absence of a prosthetic metal ion in methylamine dehydrogenase as well as its existence in the periplasm renders TTQ biogenesis more complicated, likely requiring an external enzymatic system(s).抄録全体を表示PDF形式でダウンロード (1057K) -
Masato Ohzeki, Takuro Yaoi, Hideaki Moriyama, Tairo Oshima, Nobuo Tana ...1995 年 118 巻 4 号 p. 679-680
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーIsocitrate dehydrogenase from the thermophilic bacterium, Thermus thermophilus HB8, was crystallized by the vapor diffusion hanging drop method with polyethylene glycol 6000 as the precipitant. Pillar-like crystals of about 0.6×0.6×0.3mm3 were obtained. Analysis of a series of oscillation photographs indicated that the orthorhombic crystals belonged to the I222 or I212121 space group with unit cell dimensions of a=100.1 Å, b=150.4 Å, and c=87.4 Å. Intensity data were collected up to 2.5 Å resolution.抄録全体を表示PDF形式でダウンロード (1482K) -
Shigenori Kanaya, Tsuneko Uchida1995 年 118 巻 4 号 p. 681-682
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーOn reinvestigating the primary structure of one of the two forms of ribonuclease U2, RNase U2-A, we have corrected Asp49-Gln50 in the previously reported sequence [Sato and Uchida (1975) Biochem. J. 145, 353-360; Kanaya and Uchida (1986) Biochem. J. 240, 163-170] to Glu49-Asp50.抄録全体を表示PDF形式でダウンロード (190K) -
Minako Hoshi, Michio Sato, Shunzo Kondo, Akihiko Takashima, Kaori Nogu ...1995 年 118 巻 4 号 p. 683-685
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーWe examined the subeellular distribution of two glycogen synthase kinase-3 (GSK-3) isoforms in rat cerebellum. Results from immunoelectron microscopy and subcellular fractionation revealed that one isoform, tau protein kinase I/GSK-3β (TPKI/GSK-3β), was present in mitochondria, but GSK-3α was not. Although the two GSK-3 isoforms seem to have similar properties, the difference of subcellular localization observed here suggests that TPKI/GSK-3β fulfills some specific function in mitochondria.抄録全体を表示PDF形式でダウンロード (2128K) -
Long-Sen Chang, Jordge Lin, Kou-Wha Kuo, Shinne-Ren Lin, Chun-Chang Ch ...1995 年 118 巻 4 号 p. 686-692
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーRabbits hyperimmunized with cobrotoxin from Taiwan cobra venom produced nonprecipitating as well as precipitating antibodies. Both antibody preparations exhibited higher affinity for native cobrotoxin than for reduced and S-carboxymethylated (RCM) cobrotoxin. This indicated that the epitope structures in cobrotoxin are mostly conformation-dependent. In order to identify the conformational epitopes, native cobrotoxin was hydrolyzed with acid protease A, and 12 peptides were obtained on HPLC. Three peptide fragments, AP-10, AP-11, and AP-12, showed pronounced antigenicities toward precipitating as well as non-precipitating antibodies. AP-10, AP-11, and AP-12 contained a common segment in the C-terminal region of cobrotoxin, residues 43 to 62, with intact disulfide linkages. Complete removal of the C-terminal antibodies from antisera and precipitating antibodies on a C-terminal segment-Sepharose affinity column resulted in the loss of their precipitability with cobrotoxin, whilst restoration of precipitability was observed on the addition of the C-terminal antibodies to the C-terminal antibody-depleted antisera and precipitating antibodies. Studies on the antigenic structures of RCM-cobrotoxin revealed that RCM-cobrotoxin contains an immunodominant epitope at positions 22-38. The N-terminal and C-terminal regions of RCM-cobrotoxin encompass other epitopes which exhibit low reactivities toward anti-RCM-cobrotoxin antibodies. However, no precipitated antigen-antibody complexes were observed with the mixture of anti-RCM-cobrotoxin antibodies and RCM-cobrotoxin. These results suggest that the inherently different immunogenicities with different segments might affect the precipitabilities of the resulting antibodies, and that the notable immunogenecity of the C-terminal region is related to the production of precipitating and non-precipitating antibodies against cobrotoxin.抄録全体を表示PDF形式でダウンロード (1803K) -
Masako Mizuno-Kamiya, Hiroshi Inokuchi, Yasunaga Kameyama, Koji Yashir ...1995 年 118 巻 4 号 p. 693-699
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーWe investigated the significance of the plasma membrane lipid composition in exocytosis in an in vitro interaction system using an intact secretory granular fraction (SG) isolated from the rat parotid gland. When various liposomes (as a model of plasma membranes) were added to this assay system, rapid and transient amylase release from the SG was evoked and increased by Ca2+ in a concentration-dependent manner. The extent depended upon not only the amount of liposomes but also their lipid composition. The addition of 1, 2-diacylglycerol and phosphatidic acid to egg yolk phosphatidylcholine-liposomes remarkably facilitated the release. On the other hand, that of various free fatty acids had different effects depending upon their molecular species. Furthermore, a fluorescence de-quenching study demonstrated that membrane fusion actually occurred in this interaction system, and appeared to correlate with the amylase release. These results suggest that the transient alteration of the membrane lipid composition upon cell activation is a modulator of the exocytotic membrane interaction.抄録全体を表示PDF形式でダウンロード (962K) -
Hirofumi Yura, Mitsuaki Goto, Toshihiro Akaiket1995 年 118 巻 4 号 p. 700-705
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーWe have investigated the receptor-mediated binding and uptake of low-density lipoprotein (LDL) and its insoluble complex with dextran sulfate to determine the contribution of positively charged sites in LDL to receptor-mediated interactions. Rabbit plasma LDL derivatized with FITC retained its sedimentation ability with dextran sulfate, as well as intact LDL, and binding of fluorescent LDL and its complex to liver cells was assayed by flow cytometry. Flow cytometry revealed that the binding of complex LDL with dextran sulfate, as well as that of pure LDL, increased on rat liver parenchymal cells treated with estrogen, which enhanced the expression of LDL receptors, and decreased on Hep G2 and Chang Liver cells treated with a monoclonal antibody against LDL receptors. The binding of pure LDL and complex LDL to hepatocytes was depressed by pretreatment with unlabeled LDL in a similar manner, but not with asialoglycoprotein, a ligand of asialoglycoprotein receptors on liver cells. Furthermore, we carried out a stable primary culture of rat hepatocytes, and then pure LDL and complex LDL were applied to the cultured hepatocytes. Hepatic-differentiated functions such as albumin and bile acid secretion decreased on the uptake of pure LDL and complex LDL in a similar manner. Consequently, comparative studies using pure LDL and complex LDL allowed us to determine that the complex formation with dextran sulfate had no influence on the receptor-mediated binding or uptake of LDL, and that LDL possessed binding domains for LDL receptors and sulfonic carbohydrates, containing positively charged amino acids.抄録全体を表示PDF形式でダウンロード (859K) -
Zhen-Yu Tang, Jian-Yuan Yu, Qun Zhou, Biao He, Ze-Feng Wang, Hai-Meng ...1995 年 118 巻 4 号 p. 706-709
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーThe secondary structures of native (Holo-) and Zn2+-free (Apo-) aminoacylase were examined by circular dichroism (CD) and Fourier transform-Raman (FT-Raman) spectroscopic techniques and prediction methods. Quantitative analysis of the conformationally sensitive amide I band indicates that Holo- and Apo-enzyme contain 19.3 and 17.2% helical structure, respectively. Far-UV CD spectra of Holo- and Apo-enzyme show that they contain 20.1 and 17.6% α-helix, respectively. Secondary structure prediction of aminoacylase indicates that it contains approximately 20.9% α-helical structure including 10 α-helix segments. The results show that after removal of Zn2+ in aminoacylase, the extent of ordered structure was decreased markedly. The conformation at or near the active site of aminoacylase may contain more ordered structure and the presence of Zn2+ may help to maintain the catalytically active conformation at the active site.抄録全体を表示PDF形式でダウンロード (516K) -
Masaki Kojima, Hiroshi Miyano, Ei-ichiro Suzuki, Masaru Tanokura, Kenj ...1995 年 118 巻 4 号 p. 710-716
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーTwo isozymes of ribonuclease (RNase) T1 exist in nature, i.e., Gln25 RNase T1 and Lys25 RNase T1. G1n25 RNase T1 is less stable than Lys25 RNase T1, although the enzymatic activity is not distinguishable between these two isozymes. To elucidate the effects of the replacement of Lys25 with Gln on the conformation and microenvironments of RNase T1 in detail, two-dimensional NMR spectra were measured, sequence-specific 1H NMR resonance assignments of Gln25 RNase T1 were performed, and then the determined parameters and microenvironments of Gln25 RNase T1 were compared with those of Lys25 isozyme [Hoffmann, E. and Rüterjans, H. (1988) Eur. J. Biochem. 177, 539-560]. The main chain protons were assigned for 101 out of the total of 104 amino acid residues. Secondary structure elements were identified from analysis of characteristic NOE patterns, interstrand NOE connectivities, and hydrogen-deuterium exchange rates of main chain amide protons. The results indicated that Gln25 RNase T1 contains a single α-helix and seven β-strands. The secondary structure of Gln25 RNase T1 is, thus, essentially the same as that of Lys25 RNase T1. On the other hand, comparison of the conformation-dependent shifts of Gln25 RNase T1 with those of Lys25 RNase T1 showed that the replacement of Lys25 with Gln has significant effects on the C-terminal part of the α-helix region and the base-binding site. These results may indicate that the base-binding site is relatively flexible in the RNase T1 molecule. Among the residues of the C-terminal part of the α-helix region, the protons of Asp29 were most affected in terms of their chemical shifts, which may indicate that the side chain carboxylate anion of Asp29 is the counterpart of the electrostatic interaction of Lys25 in Lys25 RNase T1. The Gln25 of Gln25 RNase T1 may have little or no interaction with Asp29, and this may be the reason why Gln25 RNase T1 is less stable than the Lys25 isozyme.抄録全体を表示PDF形式でダウンロード (1776K) -
Teruo Miyauchi, Fumie Jimma, Tadahiko Igakura, Su Yu, Masayuki Ozawa, ...1995 年 118 巻 4 号 p. 717-724
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーBasigin is a membrane glycoprotein belonging to the immunoglobulin superfamily. The mouse basigin gene was isolated from a genomic DNA library of the BALB/c mouse, and the structure of the gene and its flanking region (11.8 kb) was completely determined. The mouse basigin gene consists of seven exons and six introns spanning 7.5 kb. The distance between the first and second exons is 5.1 kb. The first immunoglobulin-like domain of the basigin molecule is encoded by the second and third exons, and the second immunoglobulin-like domain by the fourth and fifth exons. The fifth exon encodes not only the C proximal portion of the second immunoglobulin-like domain, but also the transmembrane domain and a small portion of the cytoplasmic domain. Thus, the organization of the basigin gene is unique. The 5' upstream sequence of the basigin gene contains no TATA box or CAAT box, but has a CpG-rich island. The BALB/c genomic sequence of all seven exons is consistent with the cDNA sequences of the 129/SV and Swiss mice except several minor substitutions in the 3'-terminal sequence of the 3'-noncoding region. No protein polymorphism has so far been found in basigin of different mouse strains.抄録全体を表示PDF形式でダウンロード (980K) -
Se-Ho Park, Jeong Ho Yoon, Hyun-Ah Cho, Yeong Do Kwon, Rho Hyun Seong, ...1995 年 118 巻 4 号 p. 725-733
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーA genomic DNA clone containing the 5'-end of the rat topo IIα gene was isolated and the intron/exon structure of a 4.0 kb region encompassing the translation initiation site was determined. Multiple transcription initiation sites were found at positions -128, -110, and -100 bp upstream of the ATG codon. A minimal promoter region extending from -192 to the translation initiation codon was identified on transient expression analysis. This region lacks a TATA motif, is moderately GC-rich and contains a high number of CpG dinucleotides, which is characteristic of a housekeeping gene promoter. The fragment extending to position -242 exhibited maximal promoter activity. Putative regulatory elements were delineated within and immediately upstream of the minimal promoter region. On gel retardation and DNase I footprint analyses, two regions, between positions -195 to -159 which interact with protein factor(s) were identified. The minimal promoter region of the rat topo IIα gene showed high sequence homology with that of human topo IIα. In a 250 bp region upstream of the translation initiation site, the sequence identity was about 70%. The basic structure of the regulatory region of the rat topo IIα gene was found to be similar to that of the human counterpart.抄録全体を表示PDF形式でダウンロード (4719K) -
Shingo Tajima, Ichiro Tokimitsu1995 年 118 巻 4 号 p. 734-737
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーMessenger RNA for BPAG2, a hemidesmosomal type XVIII collagen, was detected specifically in cultured keratinocytes, not in melanocytes or dermal fibroblasts, and was localized preferentially in the skin and cornea, not in aorta, lung, kidney, or liver. Modulation of BPAG2 expression during keratinocyte adhesion and growth in culture was studied. BPAG2 mRNA level in the spreading phase was greater than that in cell attaching and proliferating phases. Ascorbic acid stimulated the BPAG2 mRNA level to 2 times the control in a dose- and exposure time-dependent manner. These results suggest that BPAG2 is expressed at a maximum level in the cell spreading phase and is up-regulated by ascorbic acid.抄録全体を表示PDF形式でダウンロード (2729K) -
Kaeko Hayashi, Hiroyuki Izu, Kohei Oda, Ken-ichi Fukuhara, Masayoshi M ...1995 年 118 巻 4 号 p. 738-744
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーA unique carboxyl proteinase [EC 3. 4. 23. 33] from Pseudomonas sp. No. 101 is the first example of a prokaryotic enzyme which is insensitive to the classical inhibitor, pepstatin. The primary structure of the proteinase was determined by conventional methods. Pseudomonas carboxyl proteinase consists of 370 amino acid residues with one disulfide bond. This enzyme has no homologous sequence with any other known carboxyl proteinase, including carboxyl proteinase B from Scytalidium lignicolum, which is a pepstatininsensitive carboxyl proteinase. In addition, Pseudomonas carboxyl proteinase lacks the Asp*-Thr-Gly, Glu*-Thr-Gly, and Asp*-Thr-Ser-Gly (*indicates the catalytic residue) sequences which are known as the motif sequences around a pair of catalytic residues in carboxyl proteinases reported so far. The results strongly indicate that Pseudomonas carboxyl proteinase is a new type of carboxyl proteinase.抄録全体を表示PDF形式でダウンロード (809K) -
Shojiro Kadono, Masahiro Sakurai, Hideaki Moriyama, Mamoru Sato, Yoko ...1995 年 118 巻 4 号 p. 745-752
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーThe structures of 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus in complexes with its substrate, cofactor, and a cofactor analog were investigated by X-ray diffraction in a crystalline state and by small-angle X-ray scattering (SAXS) in solution. The structures at 2.8 Å resolution of the complexes with the substrate, 3-isopropylmalate (IPM), and with an analog of NAD, ADP-ribose, were both very close to the structure of the free enzyme, which adopts an open conformation. However, the binding of a ligand induced a small conformational change near the binding site. This result contrasts with results for NADP+-bound and isocitrate-bound isocitrate dehydrogenase (ICDH) from Escherichia coli, which adopts a closed conformation. The SAXS analysis in solution clearly showed that IPMDH without a ligand adopts two distinct intermediate conformations, between the open and closed states, upon binding of NADH and IPM respectively, and adopts a fully closed conformation when in a ternary complex with NADH and IPM together.抄録全体を表示PDF形式でダウンロード (2291K) -
Toshiya Endo, Yumiko Nakayama, Masato Nakai1995 年 118 巻 4 号 p. 753-759
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーWe have designed a fusion gene encoding a chimeric mitochondrial precursor protein (avidin fusion protein) that consists of the mitochondrial presequence followed by mouse dihydrofolate reductase, a spacer segment, and streptavidin. The avidin fusion protein synthesized in vitro formed a tetramer at the avidin moiety on incubation with biotin during or after the translation reaction. The avidin fusion protein purified from the Escherichia coli overexpresser cells also formed the tetramer on dilution from 6M urea into buffer containing biotin. In in vitro import experiments with isolated yeast mitochondria, the tetramer of the avidin fusion protein became stuck across both mitochondrial membranes, with its N-terminal dihydrofolate reductase moiety in the matrix and its C-terminal avidin moiety exposed on the mitochondrial surface. Accumulation of the translocation intermediate of the fusion protein inhibited the import of a mitochondria) precursor protein, and allowed us to estimate the number of mitochondrial import sites.抄録全体を表示PDF形式でダウンロード (5190K) -
Takeo Yamaguchi, Masaki Matsumoto, Eiji Kimoto1995 年 118 巻 4 号 p. 760-764
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーEffects of anion transport inhibitors on hemolysis of human erythrocytes at 200 MPa were examined. The degree of hemolysis was decreased by treating intact cells with 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS), 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulfonate, or bis(sulfosuccinimidyl)suberate, whereas 4, 4'-dinitrostilbene-2, 2'-disulfonate and pyridoxal 5'-phosphate (PLP) had little effect on the hemolysis. In contrast, the degree of hypotonic hemolysis increased upon treatment with anion transport inhibitors. From the relationship between the hemolysis at 200 MPa and anion transport, it was found that high-pressure-induced hemolysis was suppressed by the covalent binding of anion transport inhibitors to band 3. This idea was supported by the finding that the hemolysis at 200 MPa of trypsin-treated erythrocytes was suppressed by DIDS. Furthermore, the spectrin content in vesicles which are released from erythrocyte ghosts by dimyristoylphosphatidylcholine decreased upon DIDS labeling of band 3, but did not change upon PLP labeling. These results suggest that the interaction of the cytoplasmic domain of band 3 with spectrin, perhaps via ankyrin, is tightened by the covalent binding of bulky ligands to the exofacial domain of band 3.抄録全体を表示PDF形式でダウンロード (713K) -
Masaru Miyagi, Fumio Sakiyama, Ikunoshin Kato, Susumu Tsunasawa1995 年 118 巻 4 号 p. 771-779
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーThe complete covalent structure of porcine liver acylamino acid-releasing enzyme (AARE) [EC 3. 4. 19. 1], which catalyzes the hydrolysis of an N-terminally acylated peptide to release an N-acylamino acid, has been established. On basis of the amino acid sequence deduced from the cDNA sequence of porcine liver AARE [Mitta, M. et at. (1989) J. Biochem. 106, 548-555], sequence determination has been achieved by automated Edman degradation of peptides generated by chemical or enzymatic cleavages of the reduced and S-carboxymethylated protein. Ion-spray mass spectrometry was also successfully used to confirm the amino acid sequences of the peptides determined above and to elucidate both the N-terminal blocking group and the status of half-cystine residues of this protein. The protein consists of 732 amino acid residues, and the N-terminal methionine residue is blocked by an acetyl group. All of 18 half-cystine residues of this protein were proved to exist as cysteine residues. A serine residue reactive with diisopropyl fluorophosphate (DFP) was also identified as Ser587 by preparation of the AARE labeled with tritiated DFP followed by isolation and sequence analysis of a radioactive peptide obtained from its endoproteinase Asp-N digest.抄録全体を表示PDF形式でダウンロード (919K) -
Yoshinari Harada, Masahiko Nakamura, Akira Asano1995 年 118 巻 4 号 p. 780-790
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーIt is well known that skeletal muscle differentiation is accompanied by the appearance of many muscle-specific components and that some of these components are generated through muscle-specific alternative splicing. It is not clear, however, in what manner, including timing, the system that regulates the muscle-specific splicing reactions is constructed during the process of myogenic differentiation. We simultaneously examined the changes in several splicing patterns for the neural cell adhesion molecule (NCAM), β-tropomyosin, and M-type pyruvate kinase genes during myogenic differentiation of cultured myoblasts using the reverse transcription-polymerase chain reaction method. The NCAM glycosylphosphatidylinositol anchor form increased in preference to the transznembrane form immediately after the induction of differentiation, while the selection of NCAM MSD1 (muscle-specific domain 1) exons started and abruptly increased at about the time when cell-fusion appeared. M2-type pyruvate kinase was gradually substituted for the M1-type molecule. Skeletal muscle-type β-tropomyosin was predominantly selected even in myoblasts in the growth medium. As a result, each transcript of these genes independently showed a temporally distinctive pattern of change in isoform selection during the myogenic differentiation of C2C12 cells. These observations suggest that some independent regulation of alternative splicing reactions should occur during myogenic differentiation.抄録全体を表示PDF形式でダウンロード (6928K) -
Shizu Takeda, Kiyoshi Kita, Hideyo Miyazaki, Shunji Natori, Kazuhisa S ...1995 年 118 巻 4 号 p. 791-795
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーWe purified a mitochondrial GTP-binding protein, MTG33, from a particulate fraction of Ehrlich ascites tumor cells [Takeda, S., Sagara, Y., Kita, K., Natori, S., and Sekimizu, K. (1993) J. Biochem. 114, 684-690]. In the present work, three GTP-binding proteins, p23A, p23B, and p26, were purified from the same material. The Kd values of p23A, p23B, and p26 for GTP were 19, 6.8, and 4.0nM, respectively. Binding of [α-32P]GTP to these proteins was inhibited by GTP and GDP, but not appreciably by other nucleotides such as ATP, CTP, UTP, and GMP. p23A, p23B, and p26 hydrolyzed GTP to GDP as well as MTG33 did. Peptide mapping analyses revealed that these GTP-binding proteins share common primary structures with MTG33. The defined properties of the three proteins suggest structural and functional relations to MTG33, which is localized in mitochondria.抄録全体を表示PDF形式でダウンロード (2587K) -
Nam-Su Lee, Byung-Taek Kim, Dong-Hyun Kim, Kyoichi Kobashi1995 年 118 巻 4 号 p. 796-801
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーA novel type of sulfotransferase, arylsulfate sulfotransferase [EC 2. 8. 2. 22], was purified to homogeneity from Haemophilus K-12, a mouse intestinal bacterium. The purified enzyme (Mr 290, 000) is composed of four subunits (Mr 70, 000). The best donor substrate was 4-methylumbelliferyl sulfate, followed by β-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and α-naphthyl sulfate. The best acceptor substrate was α-naphthol, followed by phenol and resorcinol. The apparent Km for PNS using phenol as an acceptor and that for phenol using PNS as a donor substrate were determined to be 0.095 and 0.71mM, respectively. One of the reaction products, p-nitrophenol inhibited the enzyme noncompetitively with respect to PNS, but competitively with respect to a-naphthol. The Ki values of PNP for PNS and α-naphthol were 0.89 and 0.12mM, respectively. The other reaction product, α-naphthyl sulfate, inhibited the enzyme competitively with respect to PNS, but noncompetitively with respect to α-naphthol. The Ki values of α-naphthyl sulfate for PNS and for α-naphthol were 2.72 and 1.7mM. These results suggest that the sulfate transfer reaction proceeds according to a ping pong bi bi mechanism.抄録全体を表示PDF形式でダウンロード (1572K) -
Shuichi Furuta, Takashi Hashimoto1995 年 118 巻 4 号 p. 810-818
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーMitochondria isolated from rat liver were freeze-thawed and washed with 0.1M potassium phosphate, pH 7.4. Most of the 3-hydroxyacyl coenzyme A (CoA) dehydrogenase activities were removed and this mitochondrial membrane fraction could bind exogenous 3-hydroxyacyl-CoA dehydrogenase. 3-Hydroxyacyl-CoA dehydrogenase-binding protein was extracted from the washed membrane fraction with a buffer containing 2% Triton X-100 and 2% sodium taurodeoxycholate. The binding protein was purified by Ultrogel ACA 34 gel chromatography, calcium phosphate gel-cellulose chromatography and 3-hydroxyacylCoA dehydrogenase affinity chromatography. The molecular mass of the purified binding protein was estimated to be 140 kDa by gel filtration and its subunit molecular mass was determined as 60 kDa by SDS-PAGE suggesting that the protein is a homodimer. The binding protein and 3-hydroxyacyl-CoA dehydrogenase formed a complex at low ionic strength and the stoichiometry revealed that 1 mol of the binding protein bound 2 mol of 3-hydroxyacyl-CoA dehydrogenase. Purified 3-hydroxyacyl-CoA dehydrogenase-binding protein interacted with 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase but did not bind other mitochondrial β-oxidation enzymes. The pH optimum of the binding activity was from pH 6 to 7 and the binding activity was diminished by increasing the concentration of salt in the medium.抄録全体を表示PDF形式でダウンロード (1171K) -
Satoshi Tateishi, Seiichi Mori, Tatsuo Sugano, Naoko Hori, Eiko Ohtsuk ...1995 年 118 巻 4 号 p. 819-824
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーWe fractionated HeLa cell extracts by gel filtration and then micro-injected them into cells derived from the seven complementation groups (A-G) of xeroderma pigmentosum (XP). Distinct fractions that corrected the unscheduled DNA synthesis (UDS) of the complementation group XP cells were identified. The apparent molecular weights corresponding to complementation groups A, B, C, D, E, F, and G were estimated to be 80, 600, 600, 240, 100, 240, and 280 kDa, respectively. These factors were stable in the respective cell lines, the shortest half life being 16h for the XP-A and XP-G complementing factors. The fraction (80 kDa) that corrected the UDS in XP-A cells also complemented the defect of the XP-A cell extract in the incision of DNA containing a pyrimidine dimer in a cell-free system. The separated fractions will be useful for understanding the molecular nature of these factors and for assigning complementation groups of cells derived from suspected XP patients.抄録全体を表示PDF形式でダウンロード (1903K) -
Dong Luo, Nancy Mah, Mark Krantz, Kristoffer Wilde, David Wishart, Yi ...1995 年 118 巻 4 号 p. 825-831
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーSingle chain Fv fragments (scFv) derived from an antibody, MAb 174H.64 (Tru-ScintRSQTM kit, Biomira), were constructed in both orientations, i.e. Vh-linker-Vl and Vl-linker-Vh, but only the latter form could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein showed specific anti-idiotype binding activity. Additionally, the molecular graphic modeling has been used to identify a possible site for the introduction of an interchain disulfide bond in the framework region of Fv. These Cys-modifications of the sites were done using a method of PCR-mediated mutagenesis. The engineered protein (disulfide-stabilized Fv: dsFv) was expressed and tested for its binding activity. It was found that dsFv was as active as the corresponding scFv and more stable as determined by competitive radioimmunoassay.抄録全体を表示PDF形式でダウンロード (4477K) -
Naoto Oku, Nobuko Saito, Shoji Okada, Naoko Watanabe, Yoshiro Kobayash ...1995 年 118 巻 4 号 p. 832-835
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーInterleukin-1 (IL-1) is produced and released by various cells, including activated macrophages, and plays important roles in inflammation as well as immune responses. Since the precursor of IL-1 has no signal peptide, the mechanism of IL-1 release has been an enigma. To investigate the possibility of direct interaction of IL-1 with the lipid bilayer, the interleukin-1α (IL-1α)- or β (IL-1β)-induced permeability change of the liposomal membrane was determined. IL-1α, but not IL-1β, caused an increase in the permeability of liposomes composed of phosphatidylserine (PS), at neutral and acidic pHs, as demonstrated by measuring the efflux of calcein. On the other hand, liposomes composed of phosphatidylcholine (PC) showed no increase in permeability when incubated with IL-1α, suggesting the importance of acidic phospholipids in the interaction of IL-1α with the membrane. Furthermore, permeability of liposomal membrane was markedly increased by IL-1α in the presence of 1μM calcium ions, although a permeability change was observed even in the absence of calcium ions. IL-1α also induced the efflux of fluorescent dextran (average Mr of 39, 600), raising the possibility of translocation of IL-1α through the cell membrane.抄録全体を表示PDF形式でダウンロード (415K) -
Masayuki Ozawa, Kensuke Nuruki, Hironobu Toyoyama, Yoshitada Ohi1995 年 118 巻 4 号 p. 836-840
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーPlakoglobin is a member of a protein family with a repeating amino acid motif called the armadillo repeat, and is a cytoplasmic protein found in both adherens junctions and desmosomes. Little is known about its function, but it has been shown to form distinct complexes with cadherins or desmosomal cadherins. Also, plakoglobin has been shown to form a complex with APC, a tumor suppressor gene product. We have isolated a cDNA clone encoding plakoglobin by means of the polymerase chain reaction (PCR) from a human transitional carcinoma cell line. The cDNA has the same nucleotide sequence as the previously published one [Franke et al. (1989) Proc. Natl. Acad. Sci. USA 86, 4027-4031], except that it has a deletion of 120 bp. The deleted sequence encodes the fourth armadillo repeat. Southern blot analysis of genomic DNA revealed that there is a single copy of the plakoglobin gene per haploid genome. Cloning and sequencing of a genomic DNA fragment containing the 120-bp deletion and the surrounding sequences revealed that these sequences are encoded by a single exon sequence. PCR amplification of the genomic DNA fragment of the corresponding region excluded the possible presence of the 120-bp deletion in the gene. Therefore the variant form is most likely derived through alternative splicing of precursor RNA transcripts in an exon sequence.抄録全体を表示PDF形式でダウンロード (1731K) -
Itaru Nitta, Takuya Ueda, Takahiko Nojima, Kimitsuna Watanabe1995 年 118 巻 4 号 p. 841-849
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーWe demonstrate here that a high concentration (40-70%) of pyridine, an aromatic tertiary amine catalyst, is able to promote translation on ribosomes without the presence of soluble protein factors or chemical energy sources. Compared with Monro's fragment reaction [Methods Enzymol. 20, 472-481 (1971)], which reflects only the peptidyltransferase step, this novel translation system can produce polypeptides with chain lengths of at least several tens of residues depending on the template RNA. In the presence of 60% pyridine, poly (U) and poly (UC) promoted incorporation of the respective amino acids, phenylalanine and serine-leucine, twofold, whereas poly (A) promoted the incorporation of lysine by only 25%. The degrees of polymerization of phenylalanine and lysine were up to the decamer and around 40mer, respectively. In poly (UC)-dependent oligo(serine-leucine) synthesis, oligopeptides with a serine and leucine alternate sequence were the main products. This novel pyridine system evidently differs from the non-enzymatic translation system reported by Gavrilova and Spirin [FEBS Lett. 17, 324-326 (1971)]; the former system displays partial resistance toward deproteinization reagents such as SDS and proteinase K, whereas the latter system is completely sensitive.抄録全体を表示PDF形式でダウンロード (2584K) -
Itaru Nitta, Takuya Ueda, Kimitsuna Watanabe1995 年 118 巻 4 号 p. 850-854
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーWe have already reported a novel, in vitro translation system, promoted by pyridine instead of the usual protein factors and energy sources, which consists of only salt-washed ribosomes from Escherichia coli, aminoacyl-tRNA, a template RNA, Na+ and Mg2+ cations, and 40-60% pyridine [Nitta et al. (1994) J. Biochem. 115, 803-807 and the accompanying paper]. Here we show that in this system, pyridine can be replaced not only by pyridine derivatives but also by nucleobases or nucleosides, demonstrating that any compound harboring an aromatic tertiary amine within the molecule possesses such promoting activity. These compounds may serve to assist the peptide bond formation catalyzed by peptidyltransferase within ribosomes. The finding that nucleobases and nucleosides can play such a role in this reaction implies the possibility that these compounds were directly involved in the premordial translation system.抄録全体を表示PDF形式でダウンロード (3183K) -
Naoko Kojima, Shun-ichiro Kawabata, Yuichi Makinose, Norikazu Nishino, ...1995 年 118 巻 4 号 p. 855-861
発行日: 1995/10/01
公開日: 2008/11/18
ジャーナル フリーA metalloendopeptidase (MEP) isolated from rabbit liver microsomes with substrate specificity for peptides containing Arg at the P1 and P4 positions has recently proved to be identical to soluble angiotensin-binding protein present in the cytosol. Here we describe the peptide-degrading specificity of MEP, determined using various bioactive peptides and novel fluorogenic substrates for the enzyme. MEP degraded oligopeptides, including bradykinin, α-neoendorphin, bovine adrenal medulla dodecapeptide, substance P, bombesin, neurotensin, and α-endorphin, but not polypeptides such as reduced lysozyme and histone H4, hence, MEP probably belongs to the family of endo-oligopeptidases. It cleaved most preferentially at the -Phe-Ser- bond of bradykinin (kcat/Km =2.8×104M-1•s-1) but did not cleave high molecular weight and low molecular weight kininogens, the precursors of bradykinin. MEP did not cleave angiotensin I, dynorphin A 1-13, somatostatin, and luteinizing hormone-releasing hormone, some of which are good substrates for metalloendopeptidase-24.15, metalloendopeptidase-24.16, N-arginine dibasic convertase, and yeast endopeptidase-24.15 related peptidase. An active site-directed inhibitor of metalloendopeptidase-24.15, N-[1-(R, S)-carboxyl-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate also had no effects on the amidolytic activity of MEP. Based on the cleavage sites of bioactive peptides and processing sites of vitamin K-dependent proproteins, intramolecularly quenched fluorogenic peptide substrates were newly synthesized. Among the thirteen substrates used, the most reactive was 2-aminobenzoyl-Ala-Arg-Val-Arg-Arg-Ala-Asn-Ser-2, 4-dinitroanilinoethylamide (kcat/Km=9.3×105M-1•s-1). An angiotensin antagonist, [Sar1, Ala8] -angiotensin II, inhibited hydrolysis of the substrate by MEP in a competitive manner (Ki =7.6μM). MEP cleaved oligopeptides even on the carboxyl side of proline residue and these peptides are resistant to hydrolysis by the cytosol-derived proteasome, therefore MEP may participate in the catabolism of oligopeptides in the cytosol, together with other endo-oligopeptidases.抄録全体を表示PDF形式でダウンロード (846K)
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