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Junko Amano, Sachiko Sato, Ryuichiro Nishimura, Matsuto Mochizuki, Aki ...
1989 Volume 105 Issue 3 Pages
339-340
Published: 1989
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One of the characteristic features of the asparagine-linked sugar chains of human chorionic gonadotropin (hCG) is that their sialic acid residues occur exclusively as the Neu5Acα2→3Gal group. In order to determine whether this sialic acid linkage is important for the functional role played by the sugar moiety of hCG or not, isomeric hCG containing the Neu5Acα2→6Gal group was prepared and its hormonal activity
in vitro was investigated. On addition of the isomeric hCG to the culture medium of a murine Leydig tumor cell line, it was found that it shows the same hormonal activity as natural hCG.
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Masashi Kato, Hirofumi Aiba, Takeshi Mizuno
1989 Volume 105 Issue 3 Pages
341-347
Published: 1989
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Expression of the
ompF gene coding for an outer membrane protein of
Escherichia coli is regulated by a transcriptional activation mechanism that requires the
ompR gene product that acts on nucleotides located upstream of the -35 and -10 regions of the
ompF promoter. We previously demonstrated that this
cis-acting upstream sequence displays a sequence-directed curvature of the DNA helix. To characterize the structure and function of this upstream sequence, a series of deletion mutants and base-substitution mutants of the upstream sequence of the
ompF promoter were constructed, and their abilities as to OmpR-binding and activities of the
ompF promoter were examined after they had been connected to the
lacZ gene. The nucleotides extending from position -91 to -79 are essential not only for sequence-specific recognition of the
ompF promoter by the OmpR protein, but also for OmpR-dependent activation of the
ompF promoter. It was also demonstrated that the nucleotides extending from position -111 to -92 play a role in stimulation of the
ompF expression. A local structural alteration in the
ompF promoter was observed in some of the base-substitution mutants. Based on the results, the structure and function of the upstream sequence of the
ompF promoter are discussed in relation to activation of the
ompF promoter by the OmpR protein.
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Keiichi Fukuyama, Hiroshi Matsubara, Yukiteru Katsube, Lyndon J. Roger ...
1989 Volume 105 Issue 3 Pages
348-350
Published: 1989
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Crystals of the oxidized form of flavodoxin from a red alga,
Chondrus crispus, have been grown in ammonium sulfate solution by the dialysis method. The crystals belong to the orthorhombic system, space group P2
12
12
1, with unit cell dimensions of a=63.6, b=48.8, and c=56.8 Å. The asymmetric unit contains one molecule of flavodoxin. The crystals diffract X-rays to about 2.0 Å resolution and are stable to X-ray beams. The diffraction patterns changed significantly upon soaking the crystal in a solution of a platinum complex. The major heavy-atom sites in the platinum derivative crystal have been identified from the difference Patterson function calculated at 4 Å resolution.
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Tsutomu Nishida, Naoki Nishino, Masaaki Takano, Yasuyo Sekiguchi, Kazu ...
1989 Volume 105 Issue 3 Pages
351-357
Published: 1989
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A cDNA sequence coding for rat interleukin-1α (IL-1α) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1α cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-la. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1α mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1α is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1α in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of α type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in
Escherichia coli. Recombinant rat IL-1α produced in COS cells or
E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1α and IL-1β. This suggests that GIF activity is common to IL-ls derived from various sources.
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Yohtalou Tashima, Miyuki Terui, Hideaki Itoh, Hideo Mizunuma, Ryoji Ko ...
1989 Volume 105 Issue 3 Pages
358-361
Published: 1989
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Rat liver glucocorticoid receptor was partially purifed and characterized for its hormone binding using selenite. Selenite at very low concentrations irreversibly inhibited the hormone binding. The concentration for half maximal inhibition was approximately 2.8 μM. The inhibition was restored by dithiothreitol. The receptor-hormone complex became considerably insensitive to the selenite inhibition. The receptor inhibited by selenite was eluted at the same position as the native receptor from DEAE ion exchange and gel filtration columns. The results suggest that at least four sulfhydryl groups are located in the hormone binding domain of the receptor making a cluster.
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Showbu Sato, Yukari Nakada, Koyu Hon-nami, Kumiko Yasui, Akiko Shirats ...
1989 Volume 105 Issue 3 Pages
362-366
Published: 1989
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The
trpE gene of
Clostridium thermocellum, a moderately thermophilic and absolutely anaerobic bacterium, was cloned by its ability to complement growth of an
Escherichia coli tryptophan auxotroph (
trpE) deficient in anthranilate synthase I. The nucleotide sequence of
trpE and its flanking region was determined. The
trpE gene overlapped at the termination codon with a putative initiation codon of
trpG (
trp[
G]
D), as deduced from the amino acid sequence homology with anthranilate synthase II of
Serratia marcescens. S1-nuclease mapping of the
trpE transcript produced in
E. coli cells suggested that the promoter of
C. thermocellum was utilized by E. coli. The amino acid sequence of anthranilate synthase I of C. thermocellum predicted from the nucleotide sequence is more similar to that of an extremely thermophilic bacterium,
Thermus thermophilus HB8, than that of mesophilic bacteria.
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Shusei Obata, Seiichi Taguchi, Izumi Kumagai, Kin-ichiro Miura
1989 Volume 105 Issue 3 Pages
367-371
Published: 1989
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A gene for
Streptomyces subtilisin inhibitor (SSI) from
Streptomyces albogriseolus S-3253 was cloned into
E. coli plasmid pBR322 using two oligodeoxyribonucleotides corresponding to Asp68 to Pro77 and Asn99 to G1y107 of the protein, respectively. The SSI gene was localized on a 1.8-kbp BglII/SalI fragment. The nucleotide sequence of this 1.8-kbp fragment was determined by the dideoxy sequencing method. The amino acid sequence of the mature SSI coding region derived from the nucleotide sequence determination corresponded exactly to that from protein sequencing analysis. The nucleotide sequence analysis showed the presence of a putative signal peptide comprising 31 amino acids preceding the mature SSI region. The major transcriptional start point was identified to be 60 nucleotides upstream from the putative initiation codon for translation by the primer extension method. The -45 to -25 region upstream from transcriptional start point was quite homologous to that of CTC promoter of
Bacillus subtilis. The overall G+C content of this 1.8-kbp fragment was 72%. On the other hand, an extremely high G+C content (96%) was found at the third letter of codons in the SSI coding region.
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Shusei Obata, Susumu Furukubo, Izumi Kumagai, Hideo Takahashi, Kin-ich ...
1989 Volume 105 Issue 3 Pages
372-376
Published: 1989
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A secretory expression system for
Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host,
Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the
Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (
mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in
S. lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain,
S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from
S. lividans 66 contained three additional amino acid residues in the NH
2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from
S. lividans 66 were found to be identical with those of authentic SSI.
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Tetsuro Fujisawa, Tatzuo Ueki, Shozo Iida
1989 Volume 105 Issue 3 Pages
377-383
Published: 1989
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The small-angle X-ray scattering technique was used to characterize the overall structural change as well as the state of aggregation of troponin C upon binding various amount of Ca
2+ ions: in the Ca
2+-free state and at pCa 6.5 and 4.0. Under these conditions, the forward scattering intensities of troponin C are not much different from each other:
i.e., they coincide within 4%. From these intensities, the Ca
2+-facilitated dimerization of troponin C was not verified, and no appreciable aggregation of troponin C molecules was detected below pCa 4.0. Thus, the small-angle X-ray scattering profiles from troponin C solutions were analyzed assuming a monomeric molecule. The radii of gyration of troponin C were 27.8±0.3 Å, 23.8±0.2 Å, and 22.6±0.1 Å for the Ca
2+-free state and at pCa 6.5 and 4.0, respectively. The maximum dimension of the molecule decreases from 111 to 98 Å with increasing Ca
2+ concentration. These results indicate that the troponin C molecule shrinks remarkably as Ca
2+ ions bind to the high affinity sites of the molecule. Ca
2+ binding to the low affinity sites, on the other hand, leads to a less pronounced change. Following the interpretation of scattering from the dumbbell-shaped structure (Fujisawa, T., Ueki, T., Inoko, Y., & Kataoka, M. [1987]
J. Appl. Cryst. 20, 349-355), the two domains of the molecule move closer to each other. The distance between the centers of the two domains decreases from 46 to 35 Å. The conformation of the two domains also changes upon Ca
2+ binding: they become more compact, especially in the change from the Ca
2+-free state to pCa 6.5, in good agreement with an increase of CD and other spectroscopic measurements.
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Kazuhiko Sakata, Kazuya Hoshino, Hideo Nakagawa
1989 Volume 105 Issue 3 Pages
384-389
Published: 1989
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Gelatinases have been purified from the exudate in the chronic-phase (day 7) of carragee-nin-induced inflammation in rats. The day-7 exudate gelatinases gave two peaks on Sephadex G-150 gel filtration, the initial step of the purification. The molecular weights of the gelatinases corresponding to the two peaks were about 300 kDa (HMW fraction) and about 110 kDa (LMW fraction), respectively. The gelatinase in the HMW fraction has been purified to homogeneity; the purified gelatinase gave a single band corresponding to a molecular weight of 57 kDa on both SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-gelatin PAGE. On the other hand, the gelatinase purified from the LMW fraction was found to consist of three species, with molecular weights of 66, 64, and 57 kDa, as judged on SDS-gelatin PAGE. Granulation tissue-derived fibroblasts in culture mainly produced the 64-kDa species, which was converted to a 57-kDa species on treatment with 4-amino-phenylmercuric acetate, while rat macrophages and polymorphonuclear leukocytes mainly secreted the 96-kDa species. These results suggest that exudate gelatinases are largely produced by fibroblasts in granulation tissue and that they bind to exudate proteins, resulting in the formation of complexes with molecular weights of about 300 kDa and about 110 kDa. The gelatinases purified from the HMW and LMW fractions are metallo-proteinases, as judged from the results of inhibitor experiments. Both the gelatinases degraded gelatin, but showed to proteolytic activity toward α-casein or type I collagen. Type IV collagen was degraded at 35°C by the gelatinases purified from the LMW fraction but not by that from the HMW fraction.
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Jin-Yan Cui, Sadao Wakabayashi, Keishiro Wada, Keiichi Fukuyama, Hiros ...
1989 Volume 105 Issue 3 Pages
390-394
Published: 1989
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Department of Biology, Faculty of Science, Kanazawa University The amino acid sequences of the cysteinyl peptides of
Spirulina sp. glutathione reductase were determined.
Spirulina glutathione reductase was covalently bound to Thiopropyl-Sepharose 6B in the presence of 8M urea through thiol-disulfide exchange. After tryptic digestion, 4 distinct cysteinyl peptides were finally isolated from NADPH-reduced glutathi-one reductase and 2 from oxidized glutathione reductase. The amino acid sequences of the two cysteinyl peptides which could not be isolated from the oxidized glutathione reductase were very similar to those around the active site disulfide of the other flavoprotein disulfide oxidoreductases and a unique replacement of asparagine and valine by isoleucine and arginine between the two cysteine residues was found. The other two peptides isolated from both oxidized and reduced glutathione reductase also show considerable homology to the corresponding parts of human and
Escherichia coli glutathione reductases.
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Shuntaro Hara, Ichiro Kudo, Hyuen Wook Chang, Kunio Matsuta, Terumasa ...
1989 Volume 105 Issue 3 Pages
395-399
Published: 1989
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Extracellular phospholipase A
2 was purified about 1.7×10
5 fold to near homogeneity from human synovial fluid of rheumatoid arthritis by sequential use of column chromatogra-phies on heparin-Sepharose, butyl-Toyopearl, and reversed-phase HPLC. The final prepa-ration showed a single band on SDS-polyacrylamide gel electrophoresis, and its molecular mass was estimated to be approximately 13, 700 daltons. The purified enzyme had a pH optimum of 9.0 and required Ca
2+ for maximum activity. It hydrolyzed phosphatidyl-ethanolamine more effectively than phosphatidylserine and phosphatidylcholine. These properties were similar to those of an extracellular phospholipase A
2 detected in the peritoneal cavity of caseinate-treated rats.
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Akiho Yokota, Atsushi Harada, Shozaburo Kitaoka
1989 Volume 105 Issue 3 Pages
400-405
Published: 1989
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An improved method was devised to purify ribulose 1, 5-bisphosphate carboxylase/ oxygenase (RuBisCO) with high specific activity (2.1μpmol of CO
2 fixed/mg protein/min) from
Euglena gracilis Z. The purified enzyme stored at 80°C required treatment with dithiothreitol for full activity. The dithiothreitol-treated RuBisCO was activated by 12 mM NaHCO
3 and 20 mM MgCl
2, and the activated state was stable at least for 60 min in the presence of 4 mM ethylenediaminetetraacetate. The form of inorganic carbon fixed by the
Euglena enzyme was CO
2, as for the plant enzymes. The carboxylase reaction proceeded linearly with time for at least 8 min. The optimum pH for this reaction was 7.8 to 8.0. The carboxylase activity increased with increasing temperature up to 50°C. The activation energy for the carboxylation reaction was 10.0 kcal/mol. The Michaelis constants of Euglena RuBisCO were 30.9μM for CO
2, 560μM for O
2 and 10.5μM for ribulose 1, 5-bisphosphate. Mathematical comparison between the photosynthesis rate predicted from these enzymatic properties and the observed rate suggested that there is no CO
2-concentrating mechanism in
E. gracilis.
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Chul-Ho Yun, Hyoungman Kim
1989 Volume 105 Issue 3 Pages
406-411
Published: 1989
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The interactions of ovalbumin (OA) with large unilamellar vesicles (LUV) of phosphatidyl-serine (PS) and PS /phosphatidylethanolamine (PE) were studied. It was observed that OA induces aggregation, destabilization, and fusion of these LUV composed of acidic phos-pholipids at low pH levels. The fusion of LUV by OA was monitored by measuring the intermixing of internal aqueous contents of vesicles, by resonance energy transfer assay which follows the mixing of the membrane components, and by thin-sectioning electron microscopy. The pH profile of fusion was found to be similar to the pH-dependent binding of OA to the same phospholipid vesicles. Proteolytic digestion and hydrophobic labeling with dansyl chloride and photoreactive phosphatidylcholine (PC) of the OA-vesicle complex showed that a segment of OA with a molecular weight of approximately 2, 500 penetrates the bilayer. The amino acid composition of this segment indicated that it is the 291-322 fragment and not the putative signal sequence.
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Tadashi Yoshimoto, Hiroshi Tone, Takashi Honda, Kiyoshi Osatomi, Ryuji ...
1989 Volume 105 Issue 3 Pages
412-416
Published: 1989
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A plasmid pAPP1 with a 4 kbp insert at the Pstl site of pBR322, encoding aminopeptidase P gene of
Escherichia coli HB101 (Yoshimoto
et al. (1988)
J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of
E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37°C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of theenzyme was estimated to be 49, 650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn
2+.
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Hideo Shimada, Toshio Fukasawa, Yuzuru Ishimura
1989 Volume 105 Issue 3 Pages
417-422
Published: 1989
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We isolated a cDNA clone for myoglobin mRNA from fetal bovine skeletal muscle using a DNA fragment of human myoglobin exon 2 as a probe. The complete coding sequence of myoglobin as well as the 3'- and part of the 5'-nontranslatable sequences (546 and 66 basepairs, respectively) were determined. The amino acid sequence predicted from the nucleotide sequence was in agreement with that determined in the purified protein from adult bovine cardiac muscle (Han, K.-K., Dautrevaux, M., Chaila, X., & Biserte, G. [1970] Eur. J. Biochem. 16, 465-471), except for eight amino acid residues: Val-99→Ile, Ile-101→Val, Asn-122→Asp, ala-124→Gly-129→Ala, Ala-142→Met, Glu-144→Ala, and Lys-145→G1n. When the myoglobin cDNA was expressed in Saccharomyces cerevisiae under the control of the
GAL7 promoter, myoglobin was synthesized as a functionally active holoprotein which bound molecular oxygen reversibly. The amount of myoglobin reached nearly 1% of the total extractable protein in the yeast. N-terminal sequence analysis of the produced myoglobin revealed a glycine residue at the terminus, indicating that as in native muscle the N-terminal Met was removed in yeast by processing.
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Masahiro Tomono, Makoto Shiozaki, Hideo Ikeda
1989 Volume 105 Issue 3 Pages
423-428
Published: 1989
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We found that transducing phages carrying the
gal or bio regions of the
Escherichia coli genome were formed during
in vitro packaging of endogenous λ DNA. Structural analysis of the transducing phage genomes indicated that they were formed by abnormal excision of λ prophage. Formation of transducing phages was stimulated by oxolinic acid, an inhibitor of DNA gyrase, implying that DNA gyrase participates in the abnormal excision of λ prophage. When pBR322 DNA was added to the reaction mixture, transducing phages into which pBR322 had been inserted were produced at a high frequency. This reaction was also stimulated by oxolinic acid. Sequence analyses revealed that pBR322 is inserted into the sites of abnormal excision of the prophage. These results show that transducing phages can be formed by DNA gyrase-dependent illegitimate recombination in an
in vitro system and that secondary recombination takes place frequently at the site where the first recombina-tion occurs.
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Hideo Iwahashi, Hideko Morishita, Toshihiro Ishii, Ryojin Sugata, Ryo ...
1989 Volume 105 Issue 3 Pages
429-434
Published: 1989
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The effect of caffeic acid, a kind of catechol, on the Fenton reaction was examined by using the ESR spin trapping technique. Caffeic acid enhanced the formation of hydroxyl radicals in the reaction mixture, which contained caffeic acid, hydrogen peroxide, ferric chloride, EDTA, and potassium phosphate buffer. Chlorogenic acid, which is an ester of caffeic acid with quinic acid, also stimulated the formation of the hydroxyl radicals. Quinic acid did not stimulate the reaction, suggesting that the catechol moiety in chlorogenic acid is essential to the enhancement of the hydroxyl-radical formation. Indeed, other catechols and related compounds such as pyrocatechol, gallic acid, dopamine, and noradrenaline effectively stimulated the formation of the hydroxyl radicals. The above results confirm the idea that the catechol moiety is essential to the enhancement. Ferulic acid, 4-hydroxy-3-methoxybenzoic acid, and salicylic acid had no effect on the formation of the hydroxyl radicals. The results indicate that the enhancement by the catechols of the formation of hydroxyl radicals is diminished if a methyl ester is formed at the position of the hydroxyl group of the catechol. In the absence of iron chelators such as EDTA, DETAPAC, desferriox-amine, citrate, and ADP, formation of hydroxyl radicals was not detected, suggesting that chelators are essential to the reaction. The enhancement of the formation of hydroxyl radicals is presumably due to the reduction of ferric ions by the catechols. Thus, the catechols may exert deleterious effects on biological systems if chelators such as EDTA, DETAPAC, desferrioxamine, citrate, and ADP are present.
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Sachio Morimoto, Iwao Ohtsuki
1989 Volume 105 Issue 3 Pages
435-439
Published: 1989
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Ca
2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using
45Ca. The specific Ca
2+ binding to troponin C was obtained by subtracting the amount of Ca
2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca
2+ binding measurement at various Ca
2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Caz
2+ The Ca
2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca
2+ The Ca
2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca
2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca
2+ of the low-affinity sites on the troponin C molecule.
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Masayasu Kojima, Kensaku Mizuno, Kenji Kangawa, Hisayuki Matsuo
1989 Volume 105 Issue 3 Pages
440-443
Published: 1989
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In many peptide hormones and neuropeptides, the carboxy-terminal α-amide structure is essential in eliciting biological activity. Here we report the purification and characteriza-tion of an α-amidating enzyme from porcine atrium, in which a high concentration of α-amidating activity was detected. The enzyme was purified to homogeneity from the membrane fraction of porcine atria by five steps of chromatography, including an affinity chromatography using a Sepharose column coupled with a substrate, Tyr-Phe-Gly. The purified enzyme was found to be composed of a single polypeptide chain with an apparent molecular weight of 92, 000. This enzyme converted several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide α-amides.
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Tomomitsu Hatakeyama, Hideki Ohba, Nobuyuki Yamasaki, Gunki Funatsu
1989 Volume 105 Issue 3 Pages
444-448
Published: 1989
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The binding of saccharides to ricin E isolated from small castor beans was studied by equilibrium dialysis and spectroscopy. Equilibrium dialysis data indicate that ricin E has two galactose-binding sites, a high affinity site (HA-site) and a low affinity site (LA-site). The binding of specific saccharides to ricin E induces a shift of the fluorescence spectrum to shorter wavelength by 3 nm and UV-difference spectra with a maximum at 290 nm and a negative intensity around 300 nm. The interaction of ricin E with its specific saccharides was analyzed in terms of the variation of the intensity at 320 nm in the fluorescence spectrum and the magnitude of the negative intensity at 300 nm in the UV-difference spectra as functions of saccharide concentration. The results indicate that these spectroscopic changes are representative of the binding of saccharides to the LA-site, which contains a tryptophan residue. By comparing the association constants of saccharides for ricin E with those for ricin D, isolated from the large castor beans, it was found that the HA of ricin E binds saccharides with an affinity of less than one-half that of ricin D, while the saccharide-binding abilities of the LA-site of the two ricins were about the same.
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Masaru Himeno, Hiroshi Koutoku, Toyoko Ishikawa, Keitaro Kato
1989 Volume 105 Issue 3 Pages
449-456
Published: 1989
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Acid phosphatase associated with rat liver lysosomal membranes (M-APase) was purified about 4, 200-fold over the homogenate with 10% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included; preparation of lysosomal membranes, solubilization of the membranes with 1% Triton X-100, immunoaffinity chromatography, and gel filtration with FPLC equipped with a Sephacryl S-3001IR column. The molecular weight, estimated by gel filtration through TSK SW 3000G, was approximately 320K and SDS gel electropho-resis showed that the enzyme is composed of four identical subunits with an apparent molecular weight of 67K. The enzyme contains about 24.3% carbohydrate consisting of mannose, galactose, fucose,
N-acetylglucosamine,
N-acetylgalactosamine, and
N-acetyl-neuraminic acid in a molar ratio of 38:20:5:36:4:11, respectively. In addition, three soluble forms of acid phosphatase (C-APase I, II, and III) in lysosomal contents were separated from rat liver lysosomal contents with DEAE-Sephacel. These three enzymes were also purified using immunoaffinity chromatography followed by gel filtration. C-APase I, II, III, and M-APase have isoelectric points of 7.7-8.2, 6.6-7.0, 5.7-6.7, and 3.4-3.8, respectively. All four APases are sensitive to endo-β-
N-acetylglucosaminidase H. However, only C-APase III and M-APase are digestible with neuraminidase.Susceptibility of M-APase to neuraminidase in intact tritosomes was examined to study the topography of M-APase in tritosomal membranes. Neuraminidase susceptibility of M-APase was not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that the oligosaccharide chains containing sialic acid in M-APase are on the inside surface of the tritosomal membranes. The N-terminal 18 residues of M-APase and C-APase I were the same, that is: Arg-Ser-Leu-Arg-Phe-Val-Thr-Leu-Leu-Tyr-Arg-His-Gly-Asp-Arg-X-Pro-Val-. Electron microscopic immunocytochemistry clearly localized acid phosphatase, which is associated with the interior of lysosomal membranes and with flocculating materials present in the lysosomal matrix.
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Shin-ichiro Sano, Yoshihiro Matsuda, Hachiro Nakagawa
1989 Volume 105 Issue 3 Pages
457-460
Published: 1989
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A monoclonal antibody, 1D4, recognizing a novel brain-specific protein was obtained. The 1D4 antigen is regarded to be a glycoprotein because it was adsorbed on the Con A-Sepharose column used for its purification. The antiserum (polyclonal antibodies) against the 1D4 antigen was raised in a rabbit and shown to react with just the same molecules as the 1D4 monoclonal antibody did. It was used to detect the antigen in crude tissue homogenates. The molecular mass of the 1D4 antigen was estimated to be 89 kDa by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the brain homogenate. The 1D4 antigen had multiple isoelectric points, the pattern of the bands detected on isoelectric focusing gel being quite similar to that of Type B nucleoside diphosphatase of the brain. However, they are distinct, since Type B nucleoside diphos-phatase was not adsorbed by anti-1D4 antigen IgG-Sepharose 4B. The 1D4 antigen could not be detected in any of the peripheral organs or tissues tested. The 1D4 antigen was rich in the cerebrum, diencephalon, and cerebellum in the brain, and its content decreased with the distance of the region from the cerebrum. The amounts of the 1D4 antigen in the cerebrum and cerebellum increased with the respective developmental maturation. These findings suggest that the 1D4 antigen contributes to some brain-specific functions of the mature brain.
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Peng Wang, Jun Nishihata, Eiji Takabori, Kazuo Yamamoto, Satoshi Toyos ...
1989 Volume 105 Issue 3 Pages
461-466
Published: 1989
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We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987)
J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP
γS, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP
γS-binding form and assayed as to the radioactivity of the [
35S]GTP
γS-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4, 5-bisphosphate hydroly-sis. From approximately 5μg of membrane protein we obtained about 5 fig of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.
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Hidetaka Ushijima, Hitoshi Okamura, Yasuzo Nishina, Kiyoshi Shiga
1989 Volume 105 Issue 3 Pages
467-472
Published: 1989
Released on J-STAGE: November 18, 2008
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The effects of pH and ionic strength on the equilibrium constants and rate constants (binding and dissociation rate constants) between riboflavin binding protein (RBP) and flavins (riboflavin, 3-carboxymethylriboflavin [CMRF], and FMN) were studied by fluorometry. The equilibrium constant and the binding rate constant between RBP and riboflavin were pH-independent between pH 6 and 9, and both constants were also indepen-dent of the ionic strength, while the constants between RBP and CMRF or FMN were dependent on both pH and ionic strength. The dissociation rate constants between RBP and the flavins used here were not so dependent on pH and ionic strength in the pH region 6 to 9, and the patterns of pH profiles as a whole were similar to each other, although the constants for FMN were about 30-60 times larger than those for CMRF or riboflavin. RBP had lower affinity for FMN than for riboflavin in the neutral pH region, which is based on the small binding rate constant and the large dissociation rate constant for FMN. The former is due to an electrostatic repulsion force between negative net charges of RBP and the phosphate group of FMN, and the latter is due to steric interference by the phosphate group of FMN.
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Satoshi Inouye, Shigeyuki Aoyama, Toshiyuki Miyata, Frederick I. Tsuji ...
1989 Volume 105 Issue 3 Pages
473-477
Published: 1989
Released on J-STAGE: November 18, 2008
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The small, monomeric Ca
2+-binding photoprotein, aequorin, emits blue light by an intramolecular reaction when mixed with Ca
2+. The photoprotein is made up of coelenter-azine and molecular oxygen, bound noncovalently to apoaequorin (apoprotein). The chemical steps leading to light emission, involving the oxidative degradation of coelenter-azine, have been studied extensively, but little is known about the active site and how the molecule catalyzes the oxidation of coelenterazine. The three-dimensional structure of the protein has not been determined and therefore answers to these questions have remained unavailable. The present paper describes a procedure for preparing fairly large amounts of apoaequorin and aequorin for X-ray crystallographic studies. It consists of fusing the apoaequorin cDNA to the signal peptide coding sequence of the outer membrane protein A of
Escherichia coli, which is under the control of the lipoprotein promoter. When the cDNA was expressed in
E. coli, a large excess of the recombinant protein was produced and released into the culture medium. Purification of the protein was accomplished by acid precipitation and DEAE-cellulose chromatography. The procedure yielded 7.4 mg of recombinant apoaequorin with a purity greater than 95% from 200 ml of culture medium. On regeneration with coelenterazine, the recombinant aequorin was fully active with Ca
2+.
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Shigeru Kimura, Takao Nagoya, Nobuo Aoki
1989 Volume 105 Issue 3 Pages
478-483
Published: 1989
Released on J-STAGE: November 18, 2008
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Four monoclonal antibodies to human thrombomodulin were characterized. Binding of two of these antibodies was dependent on the presence of calcium ions, and approximately 5mM calcium was required for their maximum binding. These two antibodies inhibited the binding of thrombin to thrombomodulin, thereby inhibiting activation of protein C catalyzed by thrombin-thrombomodulin complex. These two antibodies bind to a major active fragment formed by limited proteolytic digestions of thrombomodulin with elastase and trypsin, suggesting that the antibodies bind to the thrombin-binding site (or its vicinity) located in the epidermal growth factor (EGF)-homology domain. One of the other calcium-independent antibodies also inhibited the binding of thrombin and the activation of protein C, but the inhibition was very weak and was observed only when the antibody was present in a molar excess over thrombomodulin. This antibody did not bind to the protease digests of thrombomodulin. Another calcium-independent antibody did not inhibit either thrombin binding or protein C activation, but bound to the active fragment of protease digests, suggesting that the antibody binds to a region other than the thrombin-binding site in the EGF-homology domain. These observations suggest that throm-bomodulin undergoes a calcium-dependent conformational change which may occur in proximity to a thrombin-binding site located in the EGF-homology domain.
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Saburo Hara, Yoshitaka Yamamura, Yoko Fujii, Tomohiro Mega, Tokuji Ike ...
1989 Volume 105 Issue 3 Pages
484-489
Published: 1989
Released on J-STAGE: November 18, 2008
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A chitinase was purified from the culture filtrate of
Streptomyces erythraeus (SE). The enzyme (SE chitinase) has a molecular weight of 30, 000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl. SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of
Micrococcus lysodeikticus. The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes. The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives. The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes.
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