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Volume 117 (1995)
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- Issue 5 Pages 929-
- Issue 4 Pages 683-
- Issue 3 Pages 465-
- Issue 2 Pages 231-
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Volume 117, Issue 3
Displaying 1-34 of 34 articles from this issue
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Makoto Irisawa, Makoto Komiyama1995 Volume 117 Issue 3 Pages 465-466
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe phosphodiester linkages in DNA and RNA are efficiently hydrolyzed through cooperation of the lanthanum(III) ion with the iron(III) or tin(IV) ion, although each of these metal ions exhibits poor activity. The conversion on the hydrolysis of adenylyl(3'-5')adenosine by the lanthanum(III)/iron(III) combination (10mM each) at pH 7.0 and 50°C for 5 min is 72 mol%, whereas the catalysis by the lanthanum or iron ion alone is only marginal. The activity of their combination in the hydrolysis of thymidylyl (3'-5') thymidine is more than 15-fold greater than the sum of the activity of each of the two metal ions.View full abstractDownload PDF (239K) -
Yasuaki Hiromasa, Yoichi Aso, Shoji Yamashita, Yoshikatsu Aikawa1995 Volume 117 Issue 3 Pages 467-470
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pyruvate dehydrogenase multienzyme complex was purified from Bacillus stearothermophilus by means of six gel-filtration column chromatographies; once on Cellulofine GCL-2000, twice on Sepharose CL-2B, and three times on Sephacryl S-500HR. The molecular size distribution of the complex was examined in detail by gel-filtration chromatography, analytical and sucrose-density ultracentrifugations, and dynamic light scattering. The complex was found to be homogeneous; a dimeric complex was undetectable even with a high concentration of protein (below 6.8mg/ml).View full abstractDownload PDF (420K) -
Yukio Morimoto, Tsunehiro Mizushima, Akiko Yagi, Nobuyuki Tanahashi, K ...1995 Volume 117 Issue 3 Pages 471-474
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEukaryotic proteasomes are multicatalytic proteinase complexes with a molecular weight of 750 kDa, containing, respectively, two copies of a hetero-heptamer of α-type subunits and one of β-type subunits, (α1-7β1-7)2. Proteasome was purified from bovine liver and crystallized into a hexagonal system with cell dimensions of α=b=121.83(2) Å, c=930.68(6) Å. A cylindrical particle size of 122 Å diameter and 155 Å height was determined from the molecular packing in a unit cell. The crystal gave diffraction spots up to at least 4.4 Å resolution, which was the minimum spacing of the camera used. The overall temperature factor of the enzyme was estimated to be in the range of 36.2 to 25.8 Å2. These results imply that the enzyme complex has a unique ordered structure comprising multisubunits with two types of hetero-heptamer. This ordered structure may facilitate highly organized cooperation of individual functions of subunits within the enzyme complex.View full abstractDownload PDF (1582K) -
Satoru Oshiro, Nobuhiko Katsura1995 Volume 117 Issue 3 Pages 475-479
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that DNAs directly applicable to restriction analyses and transformation of Escherichia coli can be extracted from fungi and yeasts by use of anhydrous hydrogen fluoride (HF) under a mild condition, 5min at 0°C [Oshiro, S., Katsura, N., Kitada, K., and Gunge, N. (1987) FEBS Lett. 220, 383-386]. In the present investigation, we examined whether this improved method is also applicable to extraction of RNA and protein from the fungus Chaetomium gracile. The RNA and protein were effectively extracted from the fungus after anhydrous hydrogen fluoride (HF) treatment for a short time (1min) at 0°C. The extracted poly (A) -enriched mRNA and proteins were fully intact: the mRNA purified by messenger-activated paper with poly (U) directed not only the incorporation of [3H] glycine into polypeptides but also the synthesis in a rabbit reticulocyte lysate cell-free system of proteins reactive to antibodies against the soluble fraction extracted from the fungus by HF. Analyses by gel filtration and polyacrylamide electrophoresis under native conditions showed that dextranase extracted from the fungus by HF under the same conditions had the same molecular weight and electrophretic mobility as the enzyme excreted into the medium. This suggests that the mRNAs and proteins extracted by this method are also applicable to protein synthesis directed in a cell-free system and to enzyme purification from a fungus insusceptible to lytic enzymes. This method provides a pure preparation of mRNA within 5 h and starting materials for protein purification wthin 1 h.View full abstractDownload PDF (2783K) -
Masahiro Iwakura, Bryan E. Jones, Jiabin Luo, C. Robert Matthews1995 Volume 117 Issue 3 Pages 480-488
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmino acid sequences in proteins can contain residues which complicate biochemical, biophysical, or protein engineering studies but which are not essential for folding or activity. Their replacement with other naturally-occurring amino acids which are not subject to such complications but which maintain essential properties of the protein is a desirable goal. A simple strategy for testing various mutants for their suitability is described for a pair of cysteine residues in dihydrofolate reductase (DHFR) from Escherichia coli. Using a reconstructed gene which preserves the amino acid sequence and introduces a variety of unique restriction sites, the cysteines at positions 85 and 152 were replaced by site-directed and cassette mutagenesis. The enzymatic activity, stability, and folding mechanism of six double mutant DHFR proteins were examined with the purpose of identifying a suitable alternative to wild type DHFR. The Cys85→Ala and Cys152→Ser double mutant DHFR was found to retain the four channel folding mechanism and have activity and stability which are comparable to the wild type enzyme. The replacement of the cysteines improved the resistance of DHFR to the irreversible loss of activity at high temperature.View full abstractDownload PDF (1028K) -
Masaya Miyazaki, Hiroaki Kodama, Ichiro Fujita, Yuhei Hamasaki, Sumio ...1995 Volume 117 Issue 3 Pages 489-494
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA series of dimeric fMLPs were synthesized by cross-linking with hydrophilic linkers, mono-, di-, tri-, and tetraethylene glycols. All of the dimers showed higher activities than monomeric fMLP in chemotaxis assays for human neutrophils. The magnitude of the activity enhancement was dependent on the linker length. On the other hand, production of superoxide anion was not as enhanced as chemotaxis. The experimental results indicate that these dimers, especially the triethylene glycol-bridged fMLP dimer, D-fMLP-G3, are selective agonists for chemotaxis. In order to distinguish between biological activities and intracellular signaling, rapid mobilization of the intracellular calcium was also measured. D-fMLP-G3 strongly enhanced the calcium mobilization. These results suggest that chemotaxis was correlated with the increase of the intracellular calcium concentration, whereas NADPH oxidase was not activated to generate superoxide by this elevation of cytoplasmic calcium caused by the dimeric ligand, D-fMLP-G3. The dimeric fMLPs might act as useful ligands for studying the intracellular signaling mechanisms of human neutrophils.View full abstractDownload PDF (765K) -
Makoto Ashiuchi, Tohru Yoshimura, Tae Kitamura, Yasushi Kawata, Jun Na ...1995 Volume 117 Issue 3 Pages 495-498
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe overexpression of the murI (glr) gene, which encodes the glutamate racemase of Escherichia coli, resulted in the formation of inclusion bodies of the enzyme, and little activity was found in the soluble fraction of the transformant cells. The coexpression of the groESL gene with murI caused an in vivo solubilization of glutamate racemase in an active form. We isolated the active enzyme and purified it effectively.View full abstractDownload PDF (1596K) -
Hajime Ishihara, James Douglas Engel, Masayuki Yamamoto1995 Volume 117 Issue 3 Pages 499-508
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the mechanisms underlying the tissue-restricted expression of GATA factor transcription, we have isolated and analyzed the genomic chicken GATA-3 (cGATA-3) locus. Structural analysis of the clones showed that the cGATA-3 gene consists of six exons which span more than 19 kb. Two trans-activating domains and two Zn finger domains of cGATA-3 were found to be encoded separately by exons 2/3 and 4/5, respectively, indicating that each functional domain of GATA-3 is encoded by a discrete exon. We have determined 1.7 kb of upstream promoter sequence and found a number of sequence motifs which match those of known transcription factor binding sites. Activities of presumptive regulatory regions of this gene were assessed by transfecting chimeric constructs into a chicken T cell line MSB-1. The results showed three features of cGATA-3 gene regulation. The basal promoter activity of the cGATA-3 gene is determined by sequences lying between -104 and -29 by of the promoter region. The upstream region containing the GATA and CACCC elements in close proximity (-1280 to -1152) appeared to act as a negative transcriptional regulator, whereas the region -1151 to -850 acts as a positive regulator. Thus, the expression of cGATA-3 gene is under complex regulation and the mode of regulation of cGATA-3 gene expression is suggested to be different from that of GATA-1 genes.View full abstractDownload PDF (6608K) -
Kohichi Hayakawa, Kazuhiro Kohama1995 Volume 117 Issue 3 Pages 509-514
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOkadaic acid, a toxin from black sponge, and microcystin-LR, a toxin from blue-green algae, were found to stimulate and inhibit, respectively, the actin-activated ATPase activity of skeletal muscle myosin. These effects were confirmed by monitoring the sliding movement of actin-filaments on myosin. This technique also enabled us to demonstrate the reversibility of these effects, a property that is essential for their use as a pharmacological tool for the analysis of the mechanochemical characteristics of muscular contraction. The sites of action of both toxins were within the myosin molecule, as demonstrated by monitoring (i) their effects on the intrinsic tryptophan fluorescence of heavy meromyosin and (ii) their ATP-dependent effects on the ATPase activity. The former effects further suggest that myosin heads are their actual sites of action, and the latter effects suggest that they interact with the ATPase active sites located within the heads.View full abstractDownload PDF (692K) -
Byong-Seok Choi, Alfred G. Redfield1995 Volume 117 Issue 3 Pages 515-520
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNuclear magnetic resonance measurements of proton exchange were performed on yeast tRNAPhe and yeast tRNAAsP, at temperatures from 20 to 45°C, in the presence of various levels of salt, phosphate, added magnesium, and pH. The dynamical changes of the tRNA molecule were interpreted, with the aid of firmly established assignments and the use of the saturation recovery technique. In tRNAPhe, the exchange rates in zero magnesium indicated early melting of the acceptor stem, tertiary structure, and D stem. However, in the presence of even low levels of magnesium the D stem remained intact up to high temperature, stabilized by a Mg2+ ion. A similar unfolding sequence was observed in tRNAAsp. The difference between these two tRNAs was the thermal behavior of the tertiary resonance U8-A14. In tRNAPhe, this base pair showed sharp rate increases between 32 and 39°C. However, in tRNAAsp, it remained intact up to 36°C and disappeared at 39°C, even if there was not important kinetic broadening. By measuring the temperature dependence of the exchange rates, we obtained an activation energy of 40-60 kcal/mol for all the imino protons of yeast tRNAPhe in zero magnesium. The same activation energy was obtained for tRNAPhe with equimolar concentration of magnesium. By investigation of the dependence of the exchange rates of these imino protons on solution conditions, we observed the transition from kop rate limiting in the absence of magnesium to kex rate limiting in the presence of magnesium.View full abstractDownload PDF (681K) -
Hiroko Tashiro, Nobuhito Sone1995 Volume 117 Issue 3 Pages 521-526
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome c oxidase of the thermophilic bacterium, PS3, was treated with trypsin. The hydrophilic domain of 26 kDa can be easily cleaved off from the hydrophobic anchor domain at the N-terminal region of subunit II, but remains attached to the rest of the enzyme upon gel-filtration in the presence of 0.2% lauroyl sarcosinate. The separation occurred in the presence of 5M urea in addition to 0.2% lauroyl sarcosinate. After relatively prolonged proteolysis, that induced severe activity decay, and subunit I fragmentation, the 26 kDa fragment of subunit II can be easily isolated from the rest, suggesting that this fragment with cytochrome c and CuA interacts with subunit I. The separated fragment showed absorption spectra due to CuA and cytochrome c. Reconstitution of the cytochrome oxidase activity occurred on addition of the 26 kDa fragment to the proper gel-filtration chromato-graphic fraction.View full abstractDownload PDF (3875K) -
Kiyohisa Mizumoto, Kenkoh Muroya, Toshimitsu Takagi, Toshiko Omata-Yam ...1995 Volume 117 Issue 3 Pages 527-534
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the mechanism of transcription and replication of Sendai virus, we developed an efficient and faithful in vitro transcription system using purified virus particles. The in vitro RNA synthesis was almost entirely dependent on the addition of eukaryotic cell extracts, including those from various cultured mammalian cells, mammalian tissues, and even from plant cells. The RNA products were almost identical to authentic mRNA species synthesized in the infected cells, in their size distribution, the presence of 3'-poly(A) tail and the presence of methylated 5'-cap structure (m7GpppAm). Ribonuclease protection experiments after annealing the in vitro RNA with viral genomic RNA (vRNA) indicated that the virion-associated RNA-dependent RNA polymerase transcribes correct regions of the RNA genome in vitro. The active component (s) that is required for Sendai virus mRNA synthesis was partially purified from bovine brain and was separated into at least two complementary fractions, one of which could be replaced by highly purified cellular tubulin. When viral ribonucleoprotein complexes were used instead of virus particles in the in vitro transcription, only Sendai virus-infected cell extracts supported mRNA synthesis, and extracts from uninfected cells or cells infected with other viruses were found to be inert. These results suggest that, in addition to the general factors which are present ubiquitously in eukaryotic cells, a factor (s) specific to Sendai virus-infection is required for Sendai virus transcription.View full abstractDownload PDF (6421K) -
Yong-Tae Kim, Tomonari Muramatsu, Kenji Takahashi1995 Volume 117 Issue 3 Pages 535-544
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have overproduced the leader peptidase from Escherichia coli in a high yield by using a T7 RNA polymerase/promoter system and purified the enzyme. This leader peptidase showed an apparent pH optimum of about 10 toward a synthetic peptide substrate, and was stable at temperatures below 40°C. Kinetic studies indicated that one of the active site residues in the enzyme has a pKa value of approximately 7.5. The enzyme was rapidly inactivated by reaction with N-bromosuccinimide (NBS). When approximately two tryptophan residues were oxidized with NBS, the activity was almost completely lost and this inactivation was markedly prevented by a substrate. These NBS-reactive tryptophan residues were identified as Trp300 and Trp310 by a peptide mapping analysis. This indicates that Trp300 and/or Trp310 are critically important for the activity of the leader peptidase. On the other hand, the enzyme was scarcely inhibited by treatment with N-acetylimidazole, iodoacetic acid, 5, 5'-dithiobis (2-nitrobenzoic acid), succinic anhydride, or 2, 4, 6-trinitro-benzenesulfonate. Diethylpyrocarbonate inhibited the enzyme; however, this inhibition did not seem to result from the modification of histidine residues. Thus, there seem to be no functionally important tyrosine, cysteine, or histidine residues or amino groups among the residues which readily react with these reagents. However, the enzyme was inactivated significantly by treatment with phenylglyoxal or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. Therefore, some of the arginine residues and the carboxyl groups appear to be important for the enzyme activity.View full abstractDownload PDF (1961K) -
Rong Zhang, Hitomi Yatsuki, Takahiro Kusakabe, Naoyuki Iwabe, Takashi ...1995 Volume 117 Issue 3 Pages 545-553
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNearly full-length cDNA clones for muscle-type and non-muscle-type aldolase mRNAs were cloned from λgt10 cDNA libraries constructed from skeletal muscle and liver mRNAs of lamprey (Entosphenus japonicus). The cDNA-M8 has 2, 240 by carrying an open reading frame of 1, 089 by which encodes 362 amino acids without the amino terminal methionine, while the cDNA-L3 is 1, 761 by in length and has an open reading frame of 1, 092 bp, which encodes 363 amino acids without the methionine. We designated the cDNA clones M8 and L3 as the muscle-type and non-muscle-type aldolase cDNAs, respectively. The entire amino acid sequences deduced from cDNA-M8 and -L3 show a high degree of identity to one another (76%) and also to vertebrate aldolases A (74-76%), B (68-70%), and C (71-76%) and Drosophila melanogaster aldolases α, β and γ (66-67%). Northern blot analyses using the 3'-noncoding sequences of cDNA-M8 and -L3 as hybridization probes indicated that the muscle-type mRNA is expressed mainly in the skeletal muscle, heart muscle, brain, and some other tissues, but probably not in liver, while the non-muscle-type mRNA is expressed mainly in the liver and also in brain and other tissues, except for the heart muscle. Phylogenetic analyses showed that both muscle-type and non-muscle-type al-dolases of lamprey resemble one another and might share a common ancestor with vertebrate aldolases A and C, but they are not direct ancestors of vertebrate aldolases.View full abstractDownload PDF (3308K) -
Minoru Tanaka, Kayoko Maeda, Kunio Nakashima1995 Volume 117 Issue 3 Pages 554-559
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChicken α- and β-enolase cDNAs have been cloned and analyzed to reveal that α- but not β-enolase has a Src-dependent phosphorylation site. The deduced amino acid sequence of the chicken α-enolase showed more than 90% homologies with those of other vertebrate α-enolases including amphibian (Xenopus laevis) α-like enolase. The chicken β-enolase, on the other hand, shares 84-85% amino acid sequence homology with mammalian β-enolases. These chicken enolases also showed more than 70% sequence identity with an insect (Drosophila melanogaster) enolase and around 60% with two yeast enolases. The amino acid sequence between residues 33 and 50 in chicken α-enolase coincided with the reported tryptic peptide sequence of rabbit β-enolase, the tyrosine residue in which was phosphorylated in vitro by Rous-sarcoma-virus tyrosine kinase. The present finding suggested that the tyrosine residue at position 44 in chicken α-enolase is the phosphorylation site by the tyrosine kinase. In chicken β-enolase, on the other hand, the corresponding tyrosine residue was found to be replaced with a histidine residue, in accordance with the previous observation that chicken β-enolase was not phosphorylated in vivo or in vitro. Northern blot analysis indicated that α-enolase mRNA can be expressed in a wide range of chicken tissues, and that the gene expression switch from α- to β-enolase occurs just after hatching in developing chicken muscle.View full abstractDownload PDF (2048K) -
Ayako Tani, Sannamu Lee, Osamu Oishi, Haruhiko Aoyagi, Motonori Ohno1995 Volume 117 Issue 3 Pages 560-565
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBactenecin 7 (Bac7), a cationic polypeptide from large granules of bovine neutrophils, exhibits antimicrobial activity mainly against Gram-negative bacteria. This peptide is characterized by high contents of Arg and Pro and by a unique sequence with an Arg-clustered region and three tandem repeats. In order to investigate the structure-function relationship of Bac7, two peptide fragments, which correspond to residues 1-17 (RRIRPRP-PRLPRPRPRP, an Arg-clustered region) and 46-59 (LPFPRPGPRPIPRP, one of three tandem repeats), respectively, were synthesized. Circular dichroism (CD) measurements of the two fragments indicated that they took particular conformations, although these were not defined. These peptides can bind to acidic phospholipid bilayers without marked conformational changes. When acidic phospholipid bilayers entrapping 5, 6-carboxyfiuo-rescein were treated with the peptides, no trace of the dye leaked out, indicating that the peptides lack the ability to disrupt lipid membranes. Fragment 1-17 showed weak antimicrobial activity against several bacteria, but fragment 46-59 was almost inactive. The present results suggest that longer fragments or the entire molecule of Bac7 may be required for full expression of antimicrobial activity and that Bac7 may manifest its activity by a bacteriostatic rather than a bacteriolytic mechanism.View full abstractDownload PDF (723K) -
Saori Ichikawa, Hideki Hatanaka, Yoriko Takeuchi, Shigeo Ohno, Fuyuhik ...1995 Volume 117 Issue 3 Pages 566-574
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe three-dimensional structure of the second cysteine-rich domain of protein kinase Cα (residues 95-159) was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based calculations. On the basis of 687 distance constraints derived from assigned nuclear Overhauser effect (NOE) connectivities, a total of 10 converged structures were obtained from 40 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding residues 1-7, 16-17, 30-34, and 55-65) is 0.55 Å for backbone atoms (N, Cα, C') and 1.07 Å for all non-hydrogen atoms. The molecular scaffold is maintained by triple-stranded and double-stranded twisted β-sheets packed against an α-helix and two independent zincs are coordinated by His8, Cys38, Cys41, Cys57 and Cys21, Cys24, His46, Cys49, respectively. It should be noted that the metal ligands from the two sites are interleaved and this is thought to be a new structural motif of a zinc finger domain. Based on the resultant structure, we propose an interaction site of the cysteine-rich domain of protein kinase C with diacylglycerols and phorbol esters.View full abstractDownload PDF (985K) -
Akiko Kita, Sabu Kasai, Kunio Miki1995 Volume 117 Issue 3 Pages 575-578
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe three-dimensional structure of a flavoprotein, FP390, purified. from a luminescent bacterium, Photobacterium phosphoreum, has been determined at 3 Å resolution by X-ray crystallography. Crystallographic refinements of the structural model have led to an R-factor of 0.24 for the intensity data between 6 to 3 Å resolution collected with synchrotron radiation. It was found that a homodimer of the FP390, molecules related by a non-crystallo-graphic 2-fold axis is comprised in the asymmetric unit. Two homodimers are arranged around a crystallographic 2-fold axis to form a tetrameric assembly. The monomer molecule of FP390, to which two molecules of the flavin cofactor (Q-flavin) are bound, consists of a seven-stranded parallel β-sheet which forms a half of the β-barrel structure and seven α-helices which surround one side of the β-barrel. We suggest that the reason why the Q-flavin sample prepared from FP390, is always a mixture of two components is connected with the fact that the monomer molecules has two flavin binding sites, at the dimer interface and at the molecular surface.View full abstractDownload PDF (480K) -
Kenzi Suzuki, Mitsuo Mizuguchi, Tomoharu Gomi, Eiji Itagaki1995 Volume 117 Issue 3 Pages 579-585
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSalicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivated by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1•min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatography as an apoprotein form and characterized. It could bind FAD with the same Kd value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. Vmax. for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitro-phenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P. putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme.View full abstractDownload PDF (800K) -
Tetsuo Adachi, Harutaka Yamada, Arao Futenma, Katsumi Kato, Kazuyuki H ...1995 Volume 117 Issue 3 Pages 586-590
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExtracellular-superoxide dismutase [EC 1.15.1.1] (EC-SOD) is a secretory, tetrameric glycoprotein. A prominent feature of EC-SOD is its affinity for heparin. This enzyme in serum is heterogeneous with regard to heparin-affinity and can be divided into five fractions (I) to (V) by heparin-HPLC, whereas fibroblast-secreted EC-SOD consists mainly of form (V). An intravenous injection of 50 i. u. of heparin/kg body weight into two healthy volunteers led to an immediate rise of serum EC-SOD level by 2.4-2.8-fold. Only form (V), which was a minor component in pre-heparin serum, was increased by the intravenous injection. The half-life of serum EC-SOD after the prompt rise was about 90min. The in vivo experiment using rats and an in vitro experiment strongly suggested the EC-SOD released into the plasma reconstituted the interaction with glycocalyx on the vascular endothelial cell surface in accordance with the elimination of heparin from the vascular system.View full abstractDownload PDF (574K) -
Susumu Ueno, Miyuki Kusaba, Kazuo Takeda, Masatomo Maeda, Masamitsu Fu ...1995 Volume 117 Issue 3 Pages 591-596
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Cys127-Cys150 disulfide-bonded loop (L1) of the Torpedo californica Na, K-ATPase β1 subunit was substituted with the corresponding loop of the rat β1, mouse β2, or pig H, K-ATPase a subunit. All the substituted mutant β subunits assembled with the Na, K-ATPase α subunit in a trypsin-resistant manner. The mutants with L1 from the Na, K-ATPase β subunit isoforms (rat β1 and mouse β2) each formed a functional complex with the Na, K-ATPase α subunit. On the other hand, the complex of the α subunit with the mutant β subunit that was substituted with the pig H, K-ATPase, β subunit L1, was inactive as to ATP hydrolysis. Ser131 and Phe148 located within L1, of the pig H, K-ATPase β subunit-substituted mutant were back-mutated to Pro131 and Arg148, respectively. The Phe148 to Arg mutation restored the ability of the mutant β subunit substituted with the H, K-ATPase β subunit L1, to form a functional complex with the α subunit. These results suggested that the Cys127-Cys150 loop of the Na, K-ATPase β1 subunit, especially Arg148, plays a critical role in the functional expression of Na, K-ATPase.View full abstractDownload PDF (1857K) -
Kiyoto Motojima, Sataro Goto1995 Volume 117 Issue 3 Pages 597-602
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of peroxisome proliferators on the levels of rat serum proteins were examined. The proliferators reduced the levels of several proteins in various ways with respect to proliferator-specificity and the time courses of the changes. The identification of three proteins by amino acid sequencing showed that they were functionally unrelated. This diversity suggests that several primary and secondary effects of the peroxisome proliferators caused the various changes in the levels of several serum proteins. Among these, apolipoprotein E was the most but transiently down-regulated by the two proliferators tested. Northern blots of the rat liver mRNA suggested that the primary step of the down-regulation was at pre-translation.View full abstractDownload PDF (4810K) -
Shigeru Takemori, Maki Yamaguchi, Naoto Yagi1995 Volume 117 Issue 3 Pages 603-608
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing a technique to obtain a detailed X-ray diffraction pattern from a single frog skinned muscle fiber with synchrotron radiation and an imaging plate, we studied the arrangement of myosin heads to which ADP and vanadate are bound. The presence of 1 mM vanadate during contraction caused trapping of ADP and vanadate on the myosin head. Both in the presence and absence of Ca2+, the intensities of the equatorial reflections indicated that most of the heads with ADP and vanadate were located close to the backbone of the thick filament. The presence of the first myosin layer-line at 43 nm-1. also suggested that the heads formed a helix around the shaft of the thick filament. Weak intensity of actin layer-lines suggested that the myosin heads were detached from the thin filament. The results suggest that the myosin-ADP-vanadate complex has a weak affinity toward actin regardless of the state of the regulatory system on the thin filament.View full abstractDownload PDF (1727K) -
Mahito Terabe, Shuichi Kojima, Seiichi Taguchi, Haruo Momose, Kin-ichi ...1995 Volume 117 Issue 3 Pages 609-613
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe determined the complete amino acid sequence of a novel subtilisin inhibitor, SIL15, which had been isolated from the culture supernatant of Streptomyces bikiniensis and shown to be a member of the Streptomyces subtilisin inhibitor (SSI) -like (SIL) protein family, and then identified its reactive site. SIL15 is composed of 113 amino acids and exists as a dimer. Compared with other SSI-family inhibitors, SIL15 was found to be unique in that it possesses a Gln residue at the P1 site of the reactive site and has two-residue insertions in two regions, one in the α1-helix and the other in the flexible loop region near the reactive site. Inhibition of subtilisin BPN' by SIL15 (inhibitor constant, 2.7×10-11M) was due to the presence of a Gln residue at the P1 site, which was well consistent with the results obtained for P1-site mutants of SSI and turkey ovomucoid domain 3.View full abstractDownload PDF (578K) -
Takehiro Kobayashi, Tetsuo Ishida, Kihachiro Horiike, Yoshiyuki Takaha ...1995 Volume 117 Issue 3 Pages 614-622
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cloned xylE gene encoding catechol 2, 3-dioxygenase (metapyrocatechase) from TOL plasmid in Pseudomonas putida mt-2 has been expressed in Escherichia coli W3110 to a level of _??_15% of the total soluble protein. Of the total iron in the crude extract, 45% was on the enzyme. The crystallized enzyme from E. coli had higher iron content (3.7 mol/mol enzyme) and specific activity (536 U/mg) than the enzyme from P. putida mt-2. However, no differences were observed in physicochemical, protein-chemical, and kinetic properties between the two enzymes. The enzyme was a homotetramer, and no changes were observed in the values of Mr (136, 000±5, 000) and Stokes radius (4.26 nm) in the concentration range from 0.36 nM to 2.8 μM, indicating that the native enzyme neither dissociated into subunits nor polymerized in this range. The catalytic center activity and the Km values for catechol and dioxygen were 278 s-1, 1.87 and 7.45 μM, respectively, at pH 7.5 and 25°C. The enzyme showed a broad substrate specificity. Among substrates, 4-methylcatechol and 4-chlorocatechol showed specificity constants (_??_200 μM-1•s-1) higher than that for catechol. Acetone and phenol derivatives competitively inhibited the activity against catechol. The relationship between specific activity and iron content was not linear, suggesting some conformational changes in the partially iron-depleted enzyme.View full abstractDownload PDF (2120K) -
James M. Aramini, Toshifumi Hiraoki, Yue Ke, Katsutoshi Nitta, Hans J. ...1995 Volume 117 Issue 3 Pages 623-628
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe high-affinity calcium-binding sites of bovine and human α-lactalbumin as well a equine lysozyme were analyzed by 113Cd NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched 113Cd2+ results in a signal at δ=-75.9 ppm corre-sponding to the metal ion bound to the lone Ca2+-binding site of the protein. A peak at virtually the identical resonance position (δ=-77.1 ppm) was observed in the analogous experiment with bovine α-lactalbumin. In addition, a signal upfield of these (δ=-94.7 ppm) was observed for 113Cd2+-substituted human α-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca2+-binding proteins. The field dependence of the 113Cd signals for all three proteins and bovine calmodulin were compared. At each field, the 113Cd signal linewidths for the α-lactalbumins and the lysozyme are somewhat broader than those observed for the EF-hand protein. In addition, the 113Cd linewidths for the lactalbumins and the lysozyme, especially bovine α-lactalbumin, increase dramatically with the square of the magnetic field strength, indicative of the presence of nuclear relaxation via chemical shift anisotropy and chemical exchange. The protein-bound 113Cd signals for the α-lactalbumins are also markedly affected by changes in the amount of K+ present, since Cd2+ and K+ can compete for occupation of the high-affinity Ca2+-site. Their linewidths also to some extent depend on the concentration of the protein itself. The 113Cd NMR results presented here provide further evidence in favor of a high degree of homology between the Ca2+-binding sites in these functionally diverse proteins, corroborating our earlier 43Ca NMR study of these diverse classes of calcium-binding proteins [Aramini et al. (1992) Biochemistry 31, 6761-6768].View full abstractDownload PDF (800K) -
Kuniyo Inouye, Shingo Izawa, Akitoshi Saito, Ben'ichiro Tonomura1995 Volume 117 Issue 3 Pages 629-634
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRegulation of the activity of neuraminidase of Streptococcus sp. (group K) was evaluated by examining the effects of alcohols on the hydrolysis of colominic acids catalyzed by the neuraminidase. Two kinds of alcohol binding site, activation and inhibition sites, were proposed to exist. Competitive inhibition was observed with alcohols smaller than polyethylene glycol #300 (average molecular weight: 300), so the inhibition site is considered to be the substrate binding site, the size of which was estimated to be 10 Å in diameter. On the contrary, polyethylene glycols larger than this size activated the enzyme by 1.5-1.8 times. The activity could be raised by binding of the polyethylene glycols to the activation site. This activation was shown to be due solely to the decrease in the Michaelis constant, Km. The smaller polyethylene glycols (#200 and #300) were also considered to bind to the activation site, although activation was not clearly observed due to compensation with inhibition. Strong substrate inhibition by colominic acid was also observed. The activity of Streptococcus neuraminidase was shown to be regulated intricately by the substrate colominic acid and alcohols contained in the reaction medium.View full abstractDownload PDF (1660K) -
Hideyuki Tomizawa, Hidenori Yamada, Katsutoshi Wada, Taiji Imoto1995 Volume 117 Issue 3 Pages 635-640
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of additives on the nonenzymatic deamidation of an Asn residue in a peptide and racemization of Asp and/or Asn in lysozyme were investigated at pH 6 and 100°C. These chemical reactions were accelerated by the addition of phosphate ions. Several salts suppressed the deamidation in the presence of phosphate ions, while the salts did not affect the deamidation in the absence of phosphate ion at pH 6 and 100°C. The results indicated that the effect of the salts was due to the suppression of phosphate catalysis. On the other hand, trifluoroethanol (TFE), which induces the conversion of random coiled polypeptides to secondary structured ones, dramatically suppressed the deamidation of an Asn residue in a peptide. The rate of deamidation in the presence of TFE was comparable to that of asparagine (free amino acid), which was very slowly deamidated. Because TFE could not suppress the deamidation of free asparagine, the suppression of the deamidation of an Asn residue in a peptide was attributed to suppression of the catalysis by the peptide bond in the carboxyl terminus. Since the inactivation of lysozyme was caused by multiple chemical reactions such as the deamidation and racemization, it was expected that the inactivation of lysozyme could be prevented by the addition of salts or TFE. Thus, it was confirmed that salts and TFE suppressed the lysozyme inactivation at pH 6 and 100°C.View full abstractDownload PDF (817K) -
Tadao Hashimoto, Yuji Yamamoto, Yukuo Yoshida, Kunio Tagawa1995 Volume 117 Issue 3 Pages 641-647
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial ATPase inhibitor binds to F1ATPase, forming an equimolar complex with the enzyme, and the binding site has been reported to be located in the β-subunit [Klein, G. et al. (1980) Biochemistry 19, 2919-2925] or at the interface between the α- and β-subunits of the enzyme [Mimura, H. et al. (1993) J. Biochem. 113, 350-354]. In the present study, bovine ATPase inhibitor as well as three peptide fragments of the inhibitor, Gly1-Asn51, Glu52-Asp84, and Lys46-Asp84, were used to examine the features of the binding of the inhibitor and F1 ATPase. Only the amino terminal fragment, Gly1-Asn51, exhibited a similar level of inhibitory activity to that of the native ATPase inhibitor. Although the other two carboxyl terminal side fragments did not exhibit any inhibitory activity, they interfered with the action of the intact ATPase inhibitor when they were pre-loaded to F1 ATPase. Since the two carboxyl fragments did not interfere with the inhibitory action of the amino fragment, it is inferred that the inhibitor interacts with F1 ATPase at its carboxyl terminal region prior to binding at the amino terminal region of the enzyme. Cross-linking experiments revealed that ATPase inhibitor bound to the α- and β-subunits of F1 ATPase, and that the amino terminal peptide preferentially bound to the α-subunit and the carboxyl terminal peptides to the β-subunit.View full abstractDownload PDF (3669K) -
Koichi Kasahara, Ushio Kikkawa1995 Volume 117 Issue 3 Pages 648-653
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of saturated fatty acids on the protein kinase C family were studied. Saturated fatty acids with carbon chain lengths of C12 to C14 activated the α-, β-, γ-, and ε-subspecies, and this activation was synergistic with that by diacylglycerol. Tridecanoic acid (C13) was most effective among the saturated fatty acids examined. Saturated fatty acids having carbon chain lengths of less than C10 were inert, and fatty acids with longer carbon chain lengths, such as C16 and C18, showed little effect. In contrast, fatty acids were almost inactive toward the δ-subspecies, and rather inhibited its activity in the presence of phosphatidyl-serine and diacylglycerol. Phorbol ester enhanced the activity of the α-, β-, and γ-subspecies when added with fatty acids; however, it suppressed the activity of the ε-subspecies in the presence of fatty acids. Saturated fatty acids having different effects on protein kinase C subspecies may be useful as tools for studying the mechanism and role of protein kinase C activation.View full abstractDownload PDF (622K) -
Tadashi Yoshimoto, Jun Tabira, Tsutomu Kabashima, Susumu Inoue, Kiyosh ...1995 Volume 117 Issue 3 Pages 654-660
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA gene coding for protease II (basic amino acid specific oligoendopeptidase) from Moraxella lacunata was cloned and expressed in Escherichia coli DH1. The transformant harboring a hybrid plasmid, pMPROII-12, with a 3.0-kbp insert at the PvuII-SacI site in pUC19, showed 23-fold higher enzyme activity than M. lacunata. The expressed enzyme from E. coli DH1/pMPROII-12 was purified by 40-80% ammonium sulfate fractionation, chromatography on DEAE-Toyopearl, and Sephadex G-150 gel filtration. The enzyme was most active at pH 6.5 and stable at pH 6.5-9.5. It had an optimum temperature of 35°C for 5min of reaction and was stable to up to 35°C for 30min at pH 7.0. Its molecular weight was estimated to be 80, 000 by SDS-PAGE and gel-filtration analyses. It enzyme was inhibited by diisopropyl fluorophosphate (DFP) and classified as a serine endoprotease. Its amino acid sequence was 38% homologous to that of the E. coli protease II. By alignment with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-534, Asp-619, and His-654. The enzyme was crystallized by the hanging drop vapor diffusion method using PEG 4000 as precipitant.View full abstractDownload PDF (1833K) -
Yousuke Seyama, Hideaki Otsuka, Kazumasa Ohashi, Berthe Vivien-Roels, ...1995 Volume 117 Issue 3 Pages 661-670
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Harderian gland of golden hamsters excretes alkyldiacylglycerol (ADG), the fatty acid and alkyl compositions of which differ between males and females. ADG in males contains mostly straight chain fatty acids, even- and odd-numbered, the major one being 15:0, while ADG in females contains iso- and anteiso-branched chain acids (34.0%). Iso-branching was found in both even- and odd-numbered acids, but anteiso-branching was found mostly in odd-numbered acids. The presence of propionic acid at the 3 position of the glycerol moiety in male ADG, and of isovaleric and 2-methylbutyric acids at the same position in female ADG was demonstrated by NMR spectrometry. Alkyl portions also exhibited sexual dimorphism in these lipids. ADG from males consisted of straight aliphatic chains, but branched chain components occupied almost half (45%) in ADG from females, and the branching resided at the iso- and anteiso-positions. The ADGs in glands from the two sexes were separated by latrobeads column chromatography into three and two subfractions, respectively. The fatty acid and alkyl compositions of these subfractions coincided with the above-mentioned results and with the behavior of the ADGs on thin-layer plates. These findings suggest that a sex hormone affects the metabolism of valine, leucine and isoleucine, and sexual dimorphism of ADGs occurs in the Harderian gland.View full abstractDownload PDF (2155K) -
Shigeru Saito, Shigeyoshi Sakakura, Masahiro Enomoto, Motohiko Ichijo, ...1995 Volume 117 Issue 3 Pages 671-676
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBoth placental and decidual tissues contained extractable HGF, the former HGF level (31.4±23.4 ng/mg total protein) being approximately 30 times the latter HGF level. When the localization of HGF protein and HGF mRNA in placental tissues was examined by immunohistological staining and in situ hybridization, HGF protein and HGF mRNA were detected in the mesenchymal cells of the placenta, but were absent in the cytotrophoblast and syncytiotrophoblast. Although c-met protein was expressed in the cytotrophoblast, this receptor was not detectable in the syncytiotrophoblast by immunohistochemical methods. c-met mRNA was detected in placental cell line (tPA30-1) and 4 choriocarcinoma cell lines (BeWo, Jar, Jeg-3, and NUC-1), but HGF mRNA was absent in these cells. When cytotrophoblast cells were cultured in a serum-free medium in the presence of HGF, their DNA synthesis was enhanced depending on the HGF concentration, although human placental lactogen secretion itself was not affected by HGF. These results demonstrated that HGF promotes the growth of the cytotrophoblast by the paracrine mechanism, although it does not serve as a placental differentiation factor.View full abstractDownload PDF (2550K) -
Yukiyasu Asano, Kiyohisa Mizumoto, Tokumi Maruyama, Akira Ishihama1995 Volume 117 Issue 3 Pages 677-682
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS8-Azido GTP (8-N3 GTP) was demonstrated to be polymerized into RNA by influenza virus-associated RNA polymerase at about one tenth the rate of GTP incorporation. The Km value for the azido analogue of GTP in primer-dependent RNA synthesis was 94 μM whereas Km for the natural substrate, GTP, was 6.7 μM. Upon exposure of a mixture of 8-N3 [α-32P] GTP and influenza virus ribonucleoprotein (RNP) complexes to ultraviolet light, the PB1 subunit of viral RNA polymerase was selectively radio-labeled. The photo-labeling of PB1 was competed strongly by GTP and to lesser extents by other nucleoside 5'-triphosphates. These results altogether support the prediction that the substrate-binding site (S site) of influenza RNA polymerase is located on the PB1 protein. In the presence of ApG primer, the 8-N3 GTP binding was reduced to about 40% level, suggesting that the GTP analogue can bind not only to the S site but also to the primer- and product-binding site (P site).View full abstractDownload PDF (1361K)
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