The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 110, Issue 6
Displaying 1-34 of 34 articles from this issue
  • Takao Ojima, Kiyoyoshi Nishita
    1991 Volume 110 Issue 6 Pages 847-850
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    A binary complex consisting of Mr 19, 000 and Mr 40, 000 components was co-purified with troponin from a crude troponin fraction of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle. This complex is incapable of conferring Ca2+-sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, rather strongly inhibiting it, but became capable on further complexing with Akazara scallop troponin-C. To examine the effects of the Mr 19, 000 and Mr 40, 000 components on the ATPase activity, they were separated from each other by CM-Toyopearl column chromatography. The Mr 19, 000 component strongly inhibited the Mg-ATPase activity of actomyosin-tropomyosin and the inhibition was reversed by further addition of the Akazara scallop troponin-C. On the other hand, the Mr 40, 000 component slightly increased it. On hybridization with the Akazara scallop troponin subunits, the Mr 19, 000 and Mr 40, 000 components were shown to be able to substitute for troponin-I and troponin-T, respectively. The amino acid compositions of the Mr 40, 000 component and troponin-T were almost identical, and those of the Mr 19, 000 component and Mr 17, 000 C-terminal fragment of the troponin-I resembled each other fairly well. From these results, it may be concluded that the Mr 19, 000-40, 000 binary complex is the troponin-I-troponin-T complex.
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  • Katsuyoshi Kaneko, Hiromi Takano-Ohmuro, Tomoyuki Iwai, Kazuhiro Koham ...
    1991 Volume 110 Issue 6 Pages 851-853
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Chicken gizzard actomyosin, containing the calmodulin-myosin light chain kinase (MLCK) system, was incubated in the presence of various concentrations of PSK, a protein-bound polysaccharide from Basidiomycetes, together with Ca2+ and Mg-ATP. The phosphoryla-tion of myosin was enhanced half-maximally by 10-4 g/ml of PSK. However, a similar concentration of PSK reduced the Mg-ATPase activity of the actomyosin. The former was brought about through stimulation of the MLCK activity and the latter through inhibition of the myosin ATPase activity.
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  • Keiichi Fukuyama, Hiroshi Matsubara, Yoshihiro Sanbongi, Tohru Kodama
    1991 Volume 110 Issue 6 Pages 854-855
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Cytochrome c552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, exhibits remarkable thermostability. The oxidized cytochrome c552 has been crystallized in an ethanol/water mixture by means of the vapor diffusion method. The crystals belong to the orthorhombic system, space group P21212, with unit cell dimensions of a=93.4 Å, b=52.9 Å, and c =32.4 Å. Most probably the asymmetric unit contains two molecules of cytochrome c552. The crystals diffract X-rays to better than 2.5 Å resolution and are stable to X-ray irradiation.
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  • Ryo Takano, Chiaki Imada, Kaeko Kamei, Saburo Hara
    1991 Volume 110 Issue 6 Pages 856-858
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    A new homologue of marinostatin, a peptide proteinase inhibitor, was isolated from marine Alteromonas sp. B-10-31 and designated as marinostatin D. Its amino acid sequence was determined to be Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Asp-Ser-Glu. The reactive site of marinostatin D was determined to be Met(3)-Arg(4) on the basis of the reversible cleavage and regeneration of the scissile bond catalyzed by TLCK-chymotrypsin.
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  • Kyoko Nagata, Norio Yoshida, Fusahiro Ogata, Manami Araki, Kosaku Noda
    1991 Volume 110 Issue 6 Pages 859-862
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, G1uSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitro-anilide substrates which have a Glu residue at the P1 position. G1uSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of G1uSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for G1uSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41mM (0.1 M Tris-HC1, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25mM (0.1M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with G1uSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.
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  • Kou-Wha Kuo, Chun-Chang Chang
    1991 Volume 110 Issue 6 Pages 863-867
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    High affinity antibodies to cobrotoxin were obtained by immunization with derivatives of glutaraldehyde (GA)-modified cobrotoxin. The derivatives completely lost lethality and binding activity to nicotinic acetylcholine receptors (nAChR), but retained the same antigenicity as cobrotoxin toward anti-cobrotoxin antibody. Owing to hyperimmunization with these low toxicity derivatives, a high affinity antibody to cobrotoxin was induced in a short period. We also showed that the derivatives of cobrotoxin may have altered local conformation, and residues which contribute to the intensity of binding between antigen and antibody may consequently be exposed. Hence, the modified derivatives have increased binding affinity to anti-cobrotoxin antibody. In addition, since high affinity antibodies prepared using the derivatives exhibit more potent binding affinity to cobrotoxin than conventional anti-cobrotoxin antibody, the specific neutralizing capacity of the high affinity antibodies is greatly increased. These results lead to the conclusion that the derivatives of GA-modified cobrotoxin have the same antigenicity as the native toxin, and can be used as immunogens for the production of high affinity antibodies to cobrotoxin.
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  • Akira Zanma, Yoh-ichi Matsumoto, Yasuhiko Masuho
    1991 Volume 110 Issue 6 Pages 868-872
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    We constructed conjugates of superoxide dismutase (SOD) and the Fc fragment of human immunoglobulin G. The lysyl residues of bovine erythrocyte Cu, Zn-SOD were covalently linked with cysteine residues of the Fc fragment using N-succinimidyl 4-(N-maleimido)- butylate as a crosslinking agent. Analysis by gel filtration and SDS-PAGE revealed that the conjugates were composed of one molecule of SOD linked with one molecule of Fc [SOD-(Fc)1] and one SOD molecule linked with several Fc molecule [SOD-(Fc)n]. The resulting SOD-Fc conjugates retained more than 90% of the enzyme activity of SOD. When those conjugates were administered intravenously to mice, the half-lives of SOD activity in the circulation were 29 and 42 h for SOD-(Fc)1 and SOD-(Fc)n, respectively, while free SOD had a half-life of 5 min. Intravenous administration of the conjugates to mice markedly repressed the increase in serum glutamic-oxaloacetic transaminase (GOT) activity induced by paraquat. These results suggest that SOD-Fc conjugates, which have long half-lives, effectively perform dismutation of superoxide radicals and may be useful for preventing tissue injury caused by hazardous oxygen metabolites.
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  • Tadashi Yoshimoto, Akio Kanatani, Taiji Shimoda, Tetsuya Inaoka, Toshi ...
    1991 Volume 110 Issue 6 Pages 873-878
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The prolyl endopeptidase [EC 3. 4. 21. 26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3. 5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified proly1 endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78, 705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38. 2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24. 5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110, 315-320].
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  • Jung-Yaw Lin, Shu-Chen Chu, Han-Chung Wu, Yih-Shou Hsieh
    1991 Volume 110 Issue 6 Pages 879-883
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21, 000 ± 1, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.
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  • Yoshio Imai, Masahiko Nakamura
    1991 Volume 110 Issue 6 Pages 884-888
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Threonine-301 of P450I1C2 was replaced by lysine via site-directed mutagenesis. The Lys-mutated P450 exhibited absorption spectra that were characteristic of the nitrogenous-ligand-bound form of P450. In the oxidized form, the Soret band was red-shifted as compared with the typical ferric low-spin form of P450 and the β band was more intense than the α band. In the reduced form, two Soret peaks were observable at 447 and 423 nm and their relative heights were dependent on pH, indicating the existence of two interconvertible states of ferrous Lys-mutated P450 which are in equilibrium. In addition, the interaction of external ligands with the P450 heme iron was profoundly inhibited both in the oxidized and reduced forms. These findings suggest that ε-amino nitrogen of Lys-301, which was introduced by amino acid substitution, occupies the 6th coordination position with the heme iron of the Lys-mutated P450, because, owing to conformation of the P450 protein, the ε -amino group may be located at just the right position for coordination as the internal 6th ligand.
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  • Shinobu Watarai, Misao Onuma, Tatsuji Yasuda
    1991 Volume 110 Issue 6 Pages 889-895
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Four kinds of anti-GD3 monoclonal antibodies, DSG-1, -2, -3, and -4, of the IgM class were obtained by the immunization of BALB/c mice with enzootic bovine leukosis tumor tissue-derived ganglioside GD3 inserted into liposomes with Salmonella minnesota R595 lipopoly-saccharides. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay and by enzyme immunostaining on thin-layer chromatography. The reactivities of the monoclonal antibodies obtained to four ganglioside GD3 variants [GD3 (NeuAc-NeuAc), GD3 (NeuAc-NeuGc), GD3 (NeuGc-NeuAc), and GD3 (NeuGc-NeuGc)] were tested. All of the monoclonal antibodies were found to react with GD3 (NeuAc-NeuAc) and GD3 (NeuAc-NeuGc) but not with GD3 (NeuGc-NeuAc) or GD3 (NeuGc-NeuGc). Furthermore, various purified glycosphingolipids were used to determine the specificity of these monoclonal antibodies. All 4 antibodies reacted only with ganglioside GD3 [GD3 (NeuAc-NeuAc) and GD3 (NeuAc-NeuGc)], but not with several gangliosides linking the Ga1NAc, Galβ1-3Ga1NAc, NeuAcα2-3Galβ1-3GalNAc, or NeuAcα2-8NeuAc α2-3Ga1β1-3GalNAc residue to the Gal moiety of ganglioside GD3 (GD2, GD1b, GT1b, or GQ1b, respectively), ganglioside GT1a having the same terminal NeuAcα2-8NeuAcα2-3Gal residue as ganglioside GD3, other gangliosides, and neutral glycosphingolipids. These findings suggest that the 4 monoclonal antibodies obtained may be specific for the epitope of NeuAc-α2-8Siaα2-3Galfβl-4Glc residue of ganglioside GD3.
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  • Yoichi Inada, Hideaki Watanabe, Kazuko Ohgi, Masachika Irie
    1991 Volume 110 Issue 6 Pages 896-904
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    In order to elucidate the structure-function relationship of RNases belonging to the RNase T2 family (base non-specific and adenylic acid-preferential RNase), an RNase of this family was purified from Trichoderma viride (RNase Trv) to give three closely adjacent bands with RNase activity on slab-gel electrophoresis in a yield of 20%. The three RNases gave single band with the same mobility on slab-gel electrophoresis after endoglycosidase F digestion. The enzymatic properties including base specificity of RNase Try were very similar to those of typical T2-family RNases such as RNase T2 from Aspergillus oryzae and RNase M from A. saitoi. The specific activity of RNase Try towards yeast RNA was about 13-fold higher than that of RNase M. The complete primary structure of RNase Try was determined by analyses of the peptides generated by digestion of reduced and carboxymeth-ylated RNase Try with Staphylococcus aureus V8 protease, lysylendopeptidase and α-chymotrypsin. The molecular weight of the protein moiety deduced from the sequence was 25, 883. The locations of 10 half-cystine residues were almost superimposable upon those of other RNases of this family. The homologies between RNase Try and RNase T2, RNase M, and RNase Rh (Rhizopus niveus) were 124, 132, and 92 residues, respectively. The sequences around three histidine residues, His52, His109, and His114, were highly conserved in these 4 RNases.
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  • Noriyuki Ishii, Hideki Taguchi, Masasuke Yoshida, Hideyuki Yoshimura, ...
    1991 Volume 110 Issue 6 Pages 905-908
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Two-dimensional crystals of functional chaperonin molecules, which are protein complexes of cpn60 and cpn10, isolated from Thermus thermophilus were prepared on a mercury surface under oxygen atmosphere and were observed by electron microscope after transferring them to carbon coated specimen grids. The crystals showed the hexagonal lattice with unit cell dimensions of a=b=12.4nm and γ=120°. The averaged image at a 3 nm resolution of the chaperonin has a doughnut-like shape which has seven peripheral masses and a central cavity. Preincubation of the chaperonin with MgATP changed the mobility in non-denaturating PAGE but did not cause distinguishable change of shape. The location of cpn10 in the chaperonin molecule is discussed.
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  • Masaki Hikida, Haruyuki Atomi, Yuki Fukuda, Akihisa Aoki, Tadashi Hish ...
    1991 Volume 110 Issue 6 Pages 909-914
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The presence of two genomic DNA regions encoding malate synthase (MS) was shown by Southern blot analysis of the genomic DNA from an n-alkane-assimilating yeast, Candida tropicalis, using a partial MS cDNA probe, in accordance with the fact that two types of partial MS cDNAs have previously been isolated. This was also confirmed by the restriction mapping of the two genes screened from the yeast λEMBL library. Nucleotide sequence analysis of the respective genomic DNAs, named MS-1 gene and MS-2 gene, revealed that both regions encoding MS had the same length of 1, 653 base pairs, corresponding to 551 amino acids (molecular mass of MS-1, 62, 448 Da; MS-2, 62, 421 Da). Although 29 nucleotide pairs differed in the sequences of the coding regions, the number of amino acid replacements was only one: 159Asn (MS-1)→159Ser (MS-2). In the 5'-flanking regions, there were replacements of four nucleotide pairs, deletion of one pair, and insertion of four pairs. In spite of the fact that two genomic genes were present and transcribed, RNA blot analysis demonstrated that only one band (about 2kb) was observable even when the carbon sources in the cultivation medium were changed. A comparison of the amino acid sequences was made with MSs of rape (Brassica napes L. ), cucumber seed, pumpkin seed, Escherichia colt, and Hansenula polymorpha. A high homology was observed among these enzymes, the results indicating that the protein structure was relatively well conserved through the evolution of the molecule. The replacement of only one amino acid residue seems to have little effect on the structure, even if there is some effect on the enzymatic activity. Since MS-1 and MS-2 genes were very similar and TATA-box sequences were also detected in the respective 5'-flanking regions, the mechanism regulating their biosynthesis was suggested to be present in the respective 5'-flanking regions.
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  • Kazuo Yamasaki, Taibo Yamamoto
    1991 Volume 110 Issue 6 Pages 915-921
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca2+-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1μM, and the low-affinity sites one of 36μM. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca2+-ATPase. These results indicate that 1 mol of Ca2+-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxyethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13μM. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca2+-ATPase. Vanadate binding to the solubilized Ca2+-ATPase was competitively inhibited by inorganic phosphate. When the detergent was removed to reconstitute SR membrane, vanadate binding again exhibited a biphasic concentration dependency curve. These results indicate that interactions between the ATPase molecules in intact SR membranes may involve the cooperative binding of vanadate to the enzyme.
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  • Kenji Akasaki, Yasunori Yamaguchi, Koji Furuno, Hiroshi Tsuji
    1991 Volume 110 Issue 6 Pages 922-927
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    We previously purified and characterized a major lysosomal membrane glycoprotein (r-lamp-1) from rat liver [Akasaki et al. (1990) Chem. Pharm. Bull. 38, 2766-2770]. The present study describes the purification of another major lysosomal membrane glycoprotein (r-lamp-2) from rat liver and compares the tissue distribution of r-lamp-1 and r-lamp-2 in rats. R-lamp-2 was purified to apparent electrophoretic homogeneity from rat liver by a simple method with a protein yield of approximately 4.0μg/g wet weight of liver. The purification procedure includes: preparation of tritosomal membranes, extraction of tritosomal membranes with Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. R-lamp-2 exhibited an Mr of 96, 000 on SDS-PAGE and had an acidic pI of <3.5. R-lamp-2 contained 52.3% carbohydrates. Its carbohydrate moieties were composed of numerous sialyl com-plex type N-linked oligosaccharides and small amounts of O-linked oligosaccharides. Both r-lamp-1 and r-lamp-2 were detected in all rat tissues examined by immunoblot analyses, while their apparent molecular weights differed among the tissues. Immunological quantitative analysis showed that the protein concentrations of r-lamp-2 were consistently lower than those of r-lamp-1 in all the tissues tested. There was a significant correlation with a regression coefficient of 0.86 in the tissue distribution between r-lamp-1 and r-lamp-2. A good correlation was also observed in the tissue distribution between acid phosphatase and r-lamp-2. These results suggested that r-lamp-1 and r-lamp-2 are constitutive proteins of lysosomal membranes and they are functionally related to each other.
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  • Masakazu Oyama
    1991 Volume 110 Issue 6 Pages 928-933
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Binding of an intrinsic agonist (cAMP) to specific receptors on the cell surface induces transmembrane signals for activation and desensitization (adaptation and down regulation) of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum. It is generally believed that dithiothreitol (DTT) induces the activation through interaction between the receptor and gradually accumulated cAMP, since DTT is known to inhibit cAMP-phosphodiesterase which degrades cAMP. In the present paper, we investigated the mechanism of activation of adenylate cyclase by the thiol-reducing agents, DTT and 2, 3-dimercapto-l-propanol (BAL). We found that BAL activated adenylate cyclase transiently even under conditions where the intrinsic agonist supersaturated the cAMP-receptors and competitively inhibited phosphodiesterase. This result is inconsistent with the generally accepted notion. We conclude that BAL has an independent effect from those of the intrinsic agonist (cAMP) and phosphodiesterase in activation of adenylate cyclase. Since BAL could induce activation just after the activation induced by a supersaturating concentration of the intrinsic agonist had ceased, the independent effect of BAL is not a simple enhancement of the cAMP-induced activation. Our result also suggests that the cAMP-induced adaptation (but not down regulation) suppresses the BAL-induced activation while BAL itself does not induce adaptation to cAMP or BAL. We propose that the thiol-reducing reagent induces or modifies the transmembrane activation signal for adenylate cyclase.
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  • Masakazu Oyama
    1991 Volume 110 Issue 6 Pages 934-938
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Binding of folic acid (an intrinsic agonist) to the cell surface receptors evokes transmembrane signals for activation and adaptation of guanylate cyclase in Dictyostelium discoideum. The activation signal activates this enzyme and then the adaptation signal terminates the activation. As a result, these two signals cooperatively induce a transient activation of guanylate cyclase. We investigated transmembrane signal transduction for guanylate cyclase using 2, 3-dimercapto-1-propanol (BAL, a thiol-reducing reagent) since BAL induces or modifies the transmembrane signal(s). We found that BAL induced prolonged or continuous activation of guanylate cyclase. Thus, the mode of the activation is drastically different (transient versus continuous) between folic acid and BAL. We also found that the BAL-induced continuous activation was not observed when the cells were stimulated with BAL + folic acid, while folic acid + BAL transiently induced more cGMP accumulation than folic acid alone. We lastly showed that K252a, a protein kinase inhibitor, enhanced both the folic acid-induced and the BAL-induced activation of guanylate cyclase. Our results suggest that BAL induces or mimics the activation signal for guanylate cyclase. The lack of termination in the BAL-induced activation suggests that BAL does not induce the adaptation signal or that the adaptation does not inhibit the BAL-induced activation. The former possibility is more likely since folic acid suppresses the BAL-induced continuous activation. The effect of K252a suggests that protein phosphorylation plays a role in suppression of guanylate cyclase.
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  • Hironobu Koga, Nobuko Mori, Hidenori Yamada, Yukio Nishimura, Kazuo To ...
    1991 Volume 110 Issue 6 Pages 939-944
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp =7.5) could be regarded as the pKa of the α-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp =5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the Nα-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH 5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at β-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.
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  • Takashi Yamane, Masanao Kobuke, Hideki Tsutsui, Takeru Toida, Atsuo Su ...
    1991 Volume 110 Issue 6 Pages 945-950
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The crystal structure of Streptomyces erythraeus trypsin (abbreviated as SET) has been determined in order to clarify the precise structure of the vicinity of the active site of serine protease and to understand its structure-function relationship. Crystals of SET were prepared at its active pH range (pH 5-10) without any inhibitors which might have affected the circumstances around the active sites. The structure model of SET was made based on the electron density map obtained by the multiple isomorphous replacement method at 3.5 Å resolution, and refined by the restrained least-squares method. The current model yields a crystallographic R-factor of 0.272 for 4, 968 reflections between 8 and 2.7 Å resolution. Though the sequence homology among SET, Streptomyces griseus trypsin and bovine trypsin, 32-37%, is not so high, their overall structures are similar to each other. Compari-son of the three molecular structures shows that: 1) the folding of the main chains of the three proteins is essentially the same though there are significant differences on the molecular surface; 2) the spatial arrangements of the catalytic triads in the three proteins are similar to each other; 3) in SET and S. griseus trypsin a short stretch of 310-helix is found through A1a56 to Thr59; His57 in this segment is one important amino acid residue involved in the active sites.
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  • Toshio Asao, Fumio Imai, Isamu Tsuji, Misao Tashiro, Kimikazu Iwami, F ...
    1991 Volume 110 Issue 6 Pages 951-955
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The amino acid sequence of a Bowman-Birk type proteinase inhibitor (FBI) from seeds of faba bean (Vicia faba L.) was determined by analysis of peptide fragments generated by reduction and S-carboxymethylation of enzymatically modified inhibitors, which were obtained from native FBI by limited proteolysis with TPCK-trypsin or TLCK-chymotryp-sin at pH 3.5. The established sequence showed that FBI is highly homologous with Vicia angustifolia inhibitor (VAI) but lacks the portion corresponding to the C-terminal 9 amino acids of VAI. The trypsin reactive-site peptide bond in FBI was also indicated to be Lys(16)-Ser(17) and the chymotrypsin reactive-site peptide bond to be Tyr(42)-Ser(43) by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin and by sequence compari-son with other Bowman-Birk type inhibitors.
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  • Takashi Saku, Hideaki Sakai, Yasuaki Shibata, Yuzo Kato, Kenji Yamamot ...
    1991 Volume 110 Issue 6 Pages 956-964
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.
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  • Michiya Masue, Kazuko Sukegawa, Tadao Orii, Takashi Hashimoto
    1991 Volume 110 Issue 6 Pages 965-970
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    N-Acetylgalactosamine-6-sulfate sulfatase from human placenta was purified 33, 600-fold using β-N-acetyl-D-galactosamine 6-sulfate-(1→4)- β-D-glucuronic acid-(1→3) -N-acetyl-D- [3H]galactosaminitol 6-sulfate as the substrate. This enzyme is an oligomer with a molecular mass of 120 kDa and consists of polypeptides of 40 and 15 kDa. The 15 kDa polypeptide is a glycoprotein. This purified protein has activities of N-acetylgalacto-samine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. Rabbit antiserum was raised against the purified protein. The antibody titrated N-acetylgalactosamine-6-sulfate sul-fatase and galactose-6-sulfate sulfatase. The size of the precursor of the enzyme is 60 kDa, as determined by cell-free translation. The optimal pH values of the N-acetylgalacto-samine-6-sulfate sulfatase and galactose-6-sulfate sulfatase activities are pH 3.8-4.0, and the Kms are 8 and 13 μM, respectively. Sulfate and phosphate ions are potent competitive inhibitors for the enzyme and their inhibition constants are 35 and 200μM, respectively. Cross-reactive materials of 40 and 15 kDa were detected by immunoblot analysis, in the placenta, liver, and normal fibroblasts, but not in fibroblasts from a patient with Morquio disease.
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  • Kenji Hirose, Yasushi Kawata, Noboru Yumoto, Masanobu Tokushige
    1991 Volume 110 Issue 6 Pages 971-975
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2- PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110μM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.
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  • Kiyofumi Maruyama
    1991 Volume 110 Issue 6 Pages 976-981
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxo-glutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a p K of 8.8. Several lines of evidence show that the inactivation is due to the modification of ε-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or α-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate β-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pl as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pl are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.
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  • Hisashi Oku, Sumihiro Hase
    1991 Volume 110 Issue 6 Pages 982-989
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The substrate specificity of neutral α-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co2+ hydrolyzed the Manα1-3 and Manα1-6 bonds from the non-reducing termini of Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβl-4GlcNAc (M5A), but hardly hydrolyzed the Manα1-2 bonds of Man9GlcNAc2. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Manβ1-4G1cNAc2. For the substrates with the GlcNAc structure at their reducing ends (Man5GlcNAc, Man6G1cNAc and Man9GlcNAc), the hydrolysis rate was remarkably increased: Man5GlcNAc was hydrolyzed 16 times faster than M5A, and Man9GlcNAc 40 times faster than Man9GlcNAc2. The enzyme did not hydrolyze Manα1-2 residue(s) linked to Manα1-3Manβ1-4GIcNAc. The end products were as follows:
    Manα1
    6 Manβ1-4GlcNAc
    3
    (Manα1-2)0_??_2Manα1
    These results suggest that oligomannose-type sugar chains with the G1cNAc structure at their reducing ends seem to be native substrates for neutral α-mannosidase and the enzyme seems to hydrolyze endo-β-N-acetylglucosaminidase digests of oligomannose-type sugar chains in the cytosol.
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  • Takashi Morita, Kenji Fukudome, Toshiyuki Miyata, Sadaaki Iwanaga
    1991 Volume 110 Issue 6 Pages 990-996
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    To investigate the function of the γ-carboxyglutamic acid (Gla) residues of factor IXa in the activation of factor X, a new species of bovine factor IXa, designated “factor IXaβ', ” and its corresponding Gla-domainless form, designated “Gla-domainless factor IXaβ', ” were prepared under controlled conditions and characterized. First, bovine factor IXaα was converted by α-chymotrypsin in the presence of calcium ions to factor IXaβ' (Mr 47, 000). Compared with factor IXaβ, factor IXaβ' had essentially identical activities towards a synthetic substrate, benzoyl-L-arginine ethylester (BAEE), towards an active site titrant, p-nitrophenyl-p'-guanidinobenzoate, and towards protein substrate, namely, factor X. Next, the Gla-rich region (residues 1-41) of the light chain was removed from factor IXaβ' by additional selective cleavage by α-chymotrypsin in the absence of calcium ions. Gla-domainless factor IXaβ' was purified to homogeneity on a column of DEAE-Sepharose CL-6B. The heavy chain was not altered by either chymotryptic digestion. Functional comparisons of the three activated forms, namely, factor IXaa, factor IXaα', and Gla-domainless factor IXaβ', with factor IXaβ revealed that all four activated forms of factor IX had one active-site residue per molecule and essentially identical specific esterase activity towards BAEE. However, the clotting activity of Gla-domainless factor IXaβ' was less than 0. 5% of that of factor IXaβ'. In the complete factor X-activating mixture (factor IXaβ', calcium ions, phospholipid, and factor VIIIa), factor X was activated more than ten thousand-fold more rapidly than it was by factor IXaβ' in the absence of phospholipids and factor VIIIa. However, no amplification in the activation of factor X by Gla-domainless factor IXaβ and calcium ions was observed, even in the presence of phospholipids and factor VIIIa. These results indicate that the Gla-domain of factor IXa is essential for the amplification of the activation of factor X by phospholipid and factor VIIIa. Factor IXaβ' and its Gla-domainless derivative prepared in this study are species of new factor IXa with which to investigate in greater detail the functional properties of Gla residues in factor IXa β.
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  • Tamo Fukamizo, Yasuo Ikeda, Tomohiro Araki, Takao Torikata, Mayumi Kur ...
    1991 Volume 110 Issue 6 Pages 997-1003
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The structure of lysozyme from guinea hen egg white (GEWL), which differs from hen egg white lysozyme (HEWL) by ten amino acid substitutions, was investigated by nuclear magnetic resonance (NMR) spectroscopy. GEWL and HEWL were very similar to each other in their tertiary structure as judged from the profile of 1H-NMR spectra, pH titration, and an N-acetylglucosamine trisaccharide [(G1cNAc)3] binding experiment. However, we have noticed several characteristics which distinguish GEWL from HEWL. The signal of Trp 108 indole N1H of GEWL was shifted upfield by about 0.3ppm when compared with that of HEWL, and its hydrogen exchange was faster than that of HEWL. The pKa values of Glu 35 estimated from the pH titration curve of Trp 108 indole N1H were different between GEWL and HEWL. From a careful examination of spectral changes caused by (G1cNAc)3 binding, the changes in the chemical shift values of Trp 28 C5H and Asn 59 αCH of GEWL were found to be slightly larger than those of HEWL. Ile 55 of HEWL is replaced by valine in GEWL. Such a replacement may affect the neighboring hydrogen bonding between the main chain C=0 of Leu 56 and Trp 108 indole N1H, resulting in a change in the microenvironment of the substrate-binding site near Trp 108.
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  • Akio Tomoda, Munehisa Arisawa, Saburo Koshimura
    1991 Volume 110 Issue 6 Pages 1004-1007
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    2-Amino-4-methylphenol was converted to a brownish yellow material by the lysates of human erythrocytes or purified human hemoglobin. The reaction proceeded oxidatively, coupled with the oxidation of hemoglobin. The major component of the brownish yellow material produced by oxidative condensation of 2-amino-4-methylphenol was identified as 3-amino-1, 4α-dihydro-4α, 8-dimethyl-2H-phenoxazin-2-one on the basis of its spectral data including NMR spectra, IR spectra, EI mass spectra, and absorption spectra. The changes in 3-amino-1, 4α-dihydro-4α, 8-dimethyl-2H-phenoxazin-2-one during incubation of purified human hemoglobin and 2-amino-4-methylphenol were analyzed spectro-photometrically and by using HPLC. The reaction mechanism involved may be similar to that of actinomycin synthase, which oxidizes 2-amino-5-methylphenol to the dihydro-phenoxazinone.
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  • Shinobu Fujii, Toshihiko Inoue, Seiji Inoue, Kiyoshi Ikeda
    1991 Volume 110 Issue 6 Pages 1008-1015
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    Phospholipases A2(PLA2s) are classified into two groups, I and II, according to differences in the polypeptide-chain length and intramolecular disulfide bondings. The hydrolysis of monodispersed 1, 2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by bovine pancreas PLA2 (Group I) was studied at 25°C and ionic strength 0.2, and the initial velocity data were analyzed by means of the Michaelis-Menten equation. The Michaelis constant, Km, was found to be practically independent of Ca2+ concentration and also of pH value. The latter result indicates no participation of the ionizable groups in the active site in the substrate binding. The pH-dependence curve of the logarithm of the catalytic center activity, kcat, obtained in the presence of a practically saturating amount of Ca2+, showed three transitions ascribable to the participation of three ionizable groups with pK values of 5.00, 8.40, and 9.50. The respective groups were tentatively assigned to the catalytic group His 48, the N-terminal α-amino group, and invariant Tyr 52, which is located in close proximity to the imidazole ring of His 48. Deprotonation of His 48 and protonation of Tyr 52 were shown to be essential to the catalysis. The importance of the ionization state of the α-amino group was also indicated. The present results are very similar to those for a Group I PLA2 from Naja naja atra venom, but are different from those for Group II PLA2s from Agkistrodon halys blomhoffli and Trimeresurus flavoviridis venoms which show a significant Ca2+-dependence of the substrate binding and do not show any participation of the α-amino group in the catalysis [Teshima et al. (1989) J. Biochem. 105, 1044-1051 and 106, 518-527].
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  • Nobuhito Sone, Yoshihisa Fujiwara
    1991 Volume 110 Issue 6 Pages 1016-1021
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The effects of aeration during bacterial growth on the proton translocating activity of the respiratory chain of B. stearothermophilus ATCC 8005, which is stable enough for measurement of the H+/O ratio by an oxygen pulse method, were examined. For endogenous and ascorbate-N, N, N', N'-tetramethyl p-phenylene diamine (TMPD) respiration, H+/O ratios of around 6 and 2 were obtained using resting cells grown under highly aerated conditions. The values were about 4 and 0 when cells were grown under limited-air conditions. Spectrophotometric and enzyme kinetical analyses revealed that both cytochrome caa3 and pigment-432 (cytochrome cao) were acting as terminal oxidases, while cytochrome b-558 (corresponding to the “cytochrome o-type oxidase” of the thermophilic bacterium PS3 in the previous paper [Sone, N., Kutoh, E., & Sato, K. (1990) J. Biochem. 107, 597-602]) was mainly serving in the cells grown under limited-air conditions. Measurement of the pH change upon ferrocytochrome c pulse with proteoliposomes reconstituted from the membrane extract of vigorously aerated cells and that of limited-air cells suggested that both cytochrome caa3 and pigment-432 (cytochrome cao) pump protons, while cytochrome b-558 does not.
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  • Tadayasu Ohkubo, Yoshio Taniyama, Masakazu Kikuchi
    1991 Volume 110 Issue 6 Pages 1022-1029
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The 15N signal assignment of human lysozyme was carried out by using 1H-1H and 1H-15HN two dimensional experiments. To solve the severe overlap problem of the NH signals, uniform labeling of the protein with 15N was introduced. The uniformly 15N labeled protein was prepared using a high-expression system of Saccharomyces cerevisiae. From the analyses of 1H and 15N NMR spectra, all of the backbone 15N signals of the molecule were assigned to each specific residue in the amino acid sequence. Recently published proton signal assignments [Redfield & Dobson (1990) Biochemistry, 29, 7201-7214] were confirmed by these complementary data. In addition, assignments were extended to side chain 15NH2 groups of asparagine and glutamine. Elements of secondary structure were deduced from the pattern of sequential and medium-range NOE connectivities. Two β-sheets and four α-helices could be identified in the protein, which were in good agreement with those determined by X-ray crystallography. The interaction between human lysozyme and its inhibitor N-acetyl-chitotriose was investigated by 15N-1H HMQC spectra. Most of the 15N-NH cross-peaks in the spectra were separated well enough to be followed during the titration experiment. Residues whose NH proton signals decrease in intensity upon complex formation, are located mainly around subsites B, C, and D. Local conformational changes were observed around the fourth helix adjacent to the cleft of human lysozyme.
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  • Kikuo Ogata, Ayumi Kurahashi, Syoji Tanaka, Hirokazu Ohsue, Kazuo Tera ...
    1991 Volume 110 Issue 6 Pages 1030-1036
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J. -P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KC1 concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the expection being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits. L5 was not detectable in Fraction B with this method. The presence of L5 in Fraction A was further indicated on immune dot blot analysis using an antibody against L5, which showed the presence of this protein also in Fraction B. These findings may indicate that 5SrRNA is probably present as a 5SrRNA-L5 protein complex in the macromolecular aminoacyl-tRNA synthetase complexes in the rat liver supernatant.
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  • Kikuo Ogata, Ayumi Kurahashi, Naoya Kenmochi, Kazuo Terao
    1991 Volume 110 Issue 6 Pages 1037-1044
    Published: 1991
    Released on J-STAGE: November 18, 2008
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    1) Rat liver 5SrRNA enhanced the activity of methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex (Fraction B) purified from a rat liver supernatant. 5SrRNA-L5 protein complexes (5SrRNP) had similar effects, whereas other ribosomal RNAs and E. colt 5SrRNA had no effect. 2) 5SrRNA increased the activity of the complex for methionine-dependent ATP-PP1 exchange. 3) 5SrRNA increased the activities of methionyl-, arginyl-, and isoleucyl-tRNA synthetases in the complex, but scarcely affected its leucyl-, lysyl-, and glutamyl-tRNA synthetase activities. 4) 5SrRNA increased the activities of the rat liver supernatant for the attachment of [35S]methionine, [3H] isoleucine, [3H]lysine, [3H]proline, [3H]threonine, [3H] tyrosine, and [3H]phenylalanine to endogenous tRNA markedly, and those for [3H]leucine, [3H]arginine, [3H]aspartic acid, and [3H]histidine slightly, but did not affect those for [3H] glutamic acid, [3H] glycine, [3H] valine, [3H] alanine, and [3H] tryptophan. 5) Preincubation of the rat liver supernatant with an antibody against Artemia salina ribosomal protein L5, that cross-reacted with the rat liver ribosomal protein L5, decreased the attachment of [35S]methionine and [3H]-isoleucine to endogenous tRNA, and 5SrRNA and 5SRNP enhanced these activities of the supernatant preincubated with antibody. On the other hand, the antibody did not affect that for [3H]alanine. Immune dot blot analysis using the antibody against L5 showed the presence of immunologically the same protein as L5 in the liver supernatant. Northern blot analysis of RNA in the immunoprecipitate prepared from the liver supernatant incubated with the antibody against L5 indicated that 5SrRNA was complexed with L5. These results together with those of previous experiments indicate that 5SrRNA, which may be present as 5SrRNA, plays a role as a positive effector of methionyl- and isoleucyl-tRNA synthetases in the macromolecular aminoacyl-tRNA synthetase complex.
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