The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 115 , Issue 6
Showing 1-29 articles out of 29 articles from the selected issue
  • Shun-ichi Nakamura, Yasutomi Nishizuka
    1994 Volume 115 Issue 6 Pages 1029-1034
    Published: 1994
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Upon stimulation of a cell surface receptor, a membrane phospholipid degradation cascade is often induced through the activation of several phospholipases, yielding various lipid metabolites such as diacylglycerol, free fatty acids, lysophospholipids, and phosphatidic acid. Several isoforms of protein kinase C are each activated distinctly by various combinations of the lipid metabolites, presumably in different subcellular compartments. The pivotal role of this enzyme family in the intracellular signaling network is beginning to emerge.
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  • Hidefumi Yoshii, Takeshi Furuta, Akira Yasunishi, Hiroshi Hirano
    1994 Volume 115 Issue 6 Pages 1035-1037
    Published: 1994
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The ratios of inclusion of d-limonene in α-, β-, and γ-cyclodextrin (CD) cavities were measured under low moisture conditions by a micro-aqueous method, and the minimum numbers of water molecules required for inclusion of d-limonene in α-, β-, and γ-CD cavities were determined by the auto-catalytic inclusion model. The minimum number of water molecules required for α-, β-, and γ-CD inclusion calculated from the proposed model were comparable with that in the CD cavity in the crystal state. The effect of ethanol upon the inclusion process was investigated, particularly with a low water content. This is the first study involving measurement of the minimum number of water molecules required for the inclusion of organic molecules (d-limonene) in the CD cavity.
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  • Hideaki Yajima, Jun Sumaoka, Sachiko Miyama, Makoto Komiyama
    1994 Volume 115 Issue 6 Pages 1038-1039
    Published: 1994
    Released: November 18, 2008
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    Adenosine 3', 5'-cyclic monophosphate (cAMP) is formed from adenosine triphosphate at pH 8 and 50°C by use of lanthanide ions. Pr3+ and La3+ are the most active. The cAMP formation is more efficient at higher pH, where the mixture is made homogeneous by the addition of β-cyclodextrin. The potential functioning of lanthanide ions as the catalytic center of an artificial adenylate cyclase is indicated.
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  • Koji Yashiro, Yasunaga Kameyama, Masako Mizuno-Kamiya, Sun-Ok Shin, Ak ...
    1994 Volume 115 Issue 6 Pages 1040-1046
    Published: 1994
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Chronic (5- and 10-day) administration of isoproterenol, an agent that induces the proliferation of salivary gland cells, produced increases in microsomal 1-acyl-sn-glycero-3-phosphate acyltransferase and 1-acyl-sn-glycero-3-phosphocholine acyltransferase activity in rat parotid glands in parallel with gland enlargement. This increased activity was reduced when the treatment was stopped, the reduction corresponding to the reduction in gland weight. There were significant correlations between lysophospholipid acyltransferase activity and gland weight, and between the activities of the two types of lysophospholipid acyltransferase. However, isoproterenol treatment did not affect any of the steps of the subsequent phospholipid N-methylation. These results suggest that the cell proliferation induced by chronic administration of isoproterenol in the parotid gland is accompanied by reversible and selective increases in microsomal lysophospholipid acyltransferases.
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  • Richard Jennemann, Carsten Gnewuch, Silke BoBlet, B. L. Bauer, Herbert ...
    1994 Volume 115 Issue 6 Pages 1047-1052
    Published: 1994
    Released: November 18, 2008
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    In a search for adjuvants of non-bacterial origin for immunization with ganglioside, we investigated whether chemical coupling to immune stimulatory protein could increase the immunogenicity of sialoglycosphingolipid. A novel method for the linkage of glycosphin-golipids, including gangliosides, to protein was established. The procedure includes lysis of the sphingoid double bond by ozone, reduction of the ozonolysis product to the aldehyde, and coupling to amino groups, either directly by reductamination, or by conjugation via a long aliphatic chain dicarboxylic acid linker. Using this method, gangliosides Gfpt1 (IV2-Fuc-, II3NeuAc-Gg4Cer), Glac2 [II3(NeuAc)2-LacCer], and Gtetl (II3NeuAc-Gg4Cer) were coupled to keyhole limpet hemocyanin (KLH), and the immunogenicity of the conjugates was tested. Immunization of mice with the KLH-ganglioside conjugates led in each case to the formation of IgG- and IgM antibodies that recognized the underivatized gangliosides, respectively. In contrast to this, mixtures of KLH and ganglioside proved ineffective for immunization. KLH-tumor-associated ganglioside conjugates may, therefore, be considered as possible vaccines in immune therapy of cancer.
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  • Sen-Fang Sui, Hua Wu, Jun Sheng, Yong Guo
    1994 Volume 115 Issue 6 Pages 1053-1057
    Published: 1994
    Released: November 18, 2008
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    Conformational changes of two types of proteins, water-soluble proteins (BSA and myoglobin) and membrane-associated protein (cytochrome c), induced by a negatively charged supported planar phospholipid monolayer were studied. The water-soluble proteins lost most of their ordered secondary structure and formed a random conformation in the initial stage of adsorption, and but in the later stage they retained more of the α-helical structure which was their main native secondary structure in solution. The membrane-associated protein, cytochrome c, showed a different conformational change in the adsorption process. In the initial stage, it showed an increase in β-structures but not random coils. In the later stage, it contained more α-helixes than that in solution.
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  • Yoh Sano, Hideo Inoue, Kanji Kajiwara, Hiroshi Urakawa, Yuzuru Hiragi
    1994 Volume 115 Issue 6 Pages 1058-1063
    Published: 1994
    Released: November 18, 2008
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    The self-assembly process of cucumber green mottle mosaic virus (CGMMV) protein and tobacco mosaic virus (TMV) protein was examined by the thermodynamic analysis of small-angle X-ray scattering (SAXS) data. Each polymerization step of the coat proteins was assumed to be specified by a single equilibrium constant, and the equilibrium constant was evaluated by fitting the size and shape of the constituents observed by SAXS to those calculated from an assumed polymerization scheme. The logarithmic plots of the equilibrium constant against the inverse of temperature were fitted with a straight line at each buffer concentration and the thermodynamic quantities were evaluated from its intercept (yielding entropy) and slope (yielding enthalpy). The enthalpy and entropy values of TMV protein were found to be independent of buffer concentration, whereas those of CGMMV protein depended strongly on buffer concentration. In the limit, as ionic strength tends to infinity, both the enthalpy and entropy values of CGMMV protein approach those of TMV protein. The higher negative surface charge of CGMMV protein is considered to be responsible for the formation of stable single-layered disks, and for the slow polymerization process even at higher temperature and higher buffer concentrations.
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  • Satoshi Miura, Shoko Miyazawa, Takashi Osumi, Takashi Hashimoto, Yukio ...
    1994 Volume 115 Issue 6 Pages 1064-1068
    Published: 1994
    Released: November 18, 2008
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    Cell-free translation products of hepatic free polysomal RNA from a clofibrate-treated rat were incubated at 26°C for 0-60 min with a post-heavy mitochondrial supernatant fraction from normal rat liver. Exogenously added proteinase K-resistant precursor and mature forms of peroxisomal 3-ketoacyl-CoA thiolase were recovered in a particulate fraction and increased with time. Both forms of thiolase cosedimented with peroxisomes, when the proteinase K-treated import reaction mixture was centrifuged in a sucrose density gradient. The in vitro import and processing of thiolase precursors, types A and B, was likewise reproduced with highly purified peroxisomes. These results strongly suggest that the precursor form of 3-ketoacyl-CoA thiolase is translocated into peroxisomes, apparently without tight coupling with proteolytic processing to the mature protein.
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  • Yasutake Shimizu, Danuta Kielar, Hiroshi Masuno, Yasuhiko Minokoshi, T ...
    1994 Volume 115 Issue 6 Pages 1069-1074
    Published: 1994
    Released: November 18, 2008
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    Glucose uptake into brown adipose tissue is enhanced directly by norepinephrine released from the sympathetic nerves. In the present study, we tried to establish culture conditions for brown adipocytes which are favorable for investigation of this unique glucose transport. Stromal-vascular cells isolated from the interscapular brown adipose tissue of newborn rats differentiated into brown adipocytes expressing the uncoupling protein, when the cells were maintained on collagen-coated dishes. These cells, however, did not show an increase in 2-[3H] deoxyglucose transport in response to insulin or norepinephrine, nor did they exhibit expression of the GLUT4 glucose transporter, whereas GLUT1 was present, as judged on Western blotting. Pre-treatment of confluent cells with dexamethasone induced a response of glucose transport to either insulin or norepinephrine, and the expression of GLUT4, together with notable accumulation of lipid droplets. The induction of GLUT4 expression by dexamethasone was dose-dependent and potentiated by insulin. These results indicate that treatment of cultured brown adipocytes with dexamethasone makes it feasible to analyze the mechanism underlying the enhancement of glucose transport induced by norepinephrine.
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  • Atsuhiko Ishida, Takako Kitani, Sachiko Okuno, Hitoshi Fujisawa
    1994 Volume 115 Issue 6 Pages 1075-1082
    Published: 1994
    Released: November 18, 2008
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    Incubation of calmodulin-dependent protein kinase II with Ca2+ and calmodulin resulted in a marked inactivation of the enzyme. Chelation of Ca2+ by EGTA or addition of calmodulin antagonists, W-7 or trifluoperazine, completely blocked this inactivation. The concentration required for the half-maximal inactivation, 127 μM, is three to four orders of magnitude higher than that for the half-maximal activation of the enzyme. The Ca2+/ calmodulin-independent activity of the proteolytic fragment of the enzyme, whose calmodulin-binding site involved in the enzyme activation was deleted, was also decreased by incubation with Ca2+ and calmodulin. These results suggest that calmodulin-dependent protein kinase II possesses a second, low-affinity calmodulin-binding site, which is distinct from the calmodulin-binding site involved in the activation of the enzyme, and that the binding of calmodulin to the second binding site causes the inactivation of the enzyme. The inactivation by Ca2+/calmodulin was temperature-dependent. The addition of both 500 μM ADP and 10 mM MgCl2 markedly protected the enzyme against the inactivation, while such a marked protection was not observed after the addition of either of the two alone. The addition of 5 μM autocamtide-2, a synthetic substrate peptide containing the amino acid sequence of the autophosphorylation site (Thr286/Thr287 in α/β, γ, and δ isoforms) lying within the autoinhibitory domain, also protected the enzyme against the inactivation by Ca2+/calmodulin, while syntide-2, another synthetic substrate peptide corresponding to a phosphorylation site of glycogen synthase, did not protect it even at a concentration as high as 304 μM.
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  • Masachika Irie, Kazuko Ohgi, Hideaki Watanabe, Masanori Iwama, Kazuo T ...
    1994 Volume 115 Issue 6 Pages 1083-1087
    Published: 1994
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    In order to elucidate the mechanism of action of Rhizopus niveus RNase Rh, we investigated the pH profiles of the kinetic parameters of RNase RNAP Rh, a derivative of RNase Rh, and its mutant enzymes, i.e., RNase RNAP Rh H104F, RNase RNAP Rh E105Q, and RNase RNAP Rh D51N. Based on comparisons of their profiles we concluded that protonation of His104 is indispensable for the enzymatic activity and Glu105 accelerates the enzymatic activity, especially at acid pH centered at pH 3.5. Based on these data and the previous data on the chemical modification and enzymatic properties of other mutant enzymes, we propose the following as a possible mechanism of RNase Rh action. (i) His109 participates in enzymatic action as a general base catalyst which removes the hydrogen of the 2'-OH of the ribose moiety. (ii) His46 participates in the reaction as a general acid catalyst which interacts with the 5'-oxygen atom of the scissile phosphodiester bond and becomes a proton donor to the departing nucleoside or nucleotide. (iii) His104 interacts with phosphate anion and its protonation is favorable for the enzymatic activity. (iv) Since the protonated form of Glu105 is more favorable for activity, we postulate two possible roles for Glu105: (a) it stabilizes the intermediate, and (b) it interacts with the oxygen atom of P=O and polarizes the phosphorus atom.
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  • Shyuichiro Matsubara, Mitsuhiko Take, Claudio Pedraza, Takashi Muramat ...
    1994 Volume 115 Issue 6 Pages 1088-1096
    Published: 1994
    Released: November 18, 2008
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    MK is a gene that is activated by retinoic acid in embryonal carcinoma (EC) cells and is expressed temporarily during the mid-gestation period of mouse embryogenesis. Midkine, the product of the gene is a novel heparin-binding growth/differentiation factor with neurite outgrowth and neurotrophic activities. The regulatory DNA element in the retinoic acid-induced expression of the MK gene has been investigated. The 1.9 kb 5'-flanking region of the MK gene can mediate retinoic acid-responsive gene expression in F9 and HM-1 EC cells. Analysis of this region by deletion mutagenesis in F9 EC cells shows that there is a retinoic acid-responsive enhancer (designated as REM1) around 900 by upstream from the transcription start site. This enhancer is composed of two sequence elements, which are located between -1006 and -895 and between -901 and -794. The core element of the upstream region (-971 to -955), whose deletion abolished the retinoic acid responsiveness, contained a sequence highly homologous to a binding site for retinoic acid receptors. Binding of a retinoic acid receptor heterodimer to this core element was verified by gel shift assay. Thus, retinoic acid and the receptor complex can directly induce the expression of a growth/differentiation factor gene.
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  • Lisa Konishi-Imamura, Keiji Dohi, Makoto Sato, Kyoichi Kobashi
    1994 Volume 115 Issue 6 Pages 1097-1100
    Published: 1994
    Released: November 18, 2008
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    Arylsulfate sulfotransferase (ASST) from a human intestinal bacterium stoichiometrically catalyzed the transfer of the sulfate group of phenylsulfate esters to phenolic compounds. Polyclonal antibodies against ASST were obtained from rabbit sera. These antisera did not inhibit ASST activity. ASST was recognized by the IgG fraction of the antisera, but rat liver phenol sulfotransferase did not show cross-reactivity to ASST on Western blot (immunoblot) analysis. The ASST was purified by an anti-ASST immobilized affinity column chromatography to homogeneity on SDS-PAGE. The NH2-terminal amino acid and partial sequence of the purified enzyme were serine and SVKYSFEDHIINRQYEAEQAMLAKF, respectively. We corrected the previous result that the NH2-terminal of ASST was arginine.
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  • Satoko Isemura, Eiichi Saitoh
    1994 Volume 115 Issue 6 Pages 1101-1106
    Published: 1994
    Released: November 18, 2008
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    cDNA coding for the human salivary proline-rich peptide P-B was amplified by polymerase chain reaction using human submaxillary gland cDNA as the template and d (deoxy)T25 as the primer for both strands. The amplified cDNA of 0.8 kbp encodes a hydrophobic signal sequence of 22 amino acid residues and the complete sequence of P-B (57 amino acids). The determined nucleotide sequence of P-B cDNA indicates that P-B is a mature full-size protein and not the enzymic degradation product of a larger protein. The gene coding for P-B is not a member of the proline-rich protein gene family characterized previously.
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  • Atsushi Nii, Hideaki Morishita, Torn Yamakawa, Tomokazu Matsusue, Jiro ...
    1994 Volume 115 Issue 6 Pages 1107-1112
    Published: 1994
    Released: November 18, 2008
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    The second domain (R-020) of human urinary trypsin inhibitor (UTI) exerts similar inhibitory activities on trypsin, α-chymotrypsin, leukocyte elastase, and plasmin to those of UTI itself, and additionally inhibits coagulation factor Xa (FXa) and plasma kallikrein, on both of which UTI has no inhibitory effect. In the present study, we attempted to increase this FXa-inhibitory activity by modeling the structure of R-020-FXa complex and substituting one or two amino acids in R-020 using recombinant DNA technology. Molecular modeling of R-020 and FXa was performed with reference to X-ray analysis of the complex of bovine pancreatic trypsin inhibitor (BPTI) and bovine trypsin to determine the site of amino acid modification. The expression plasmids into which R-020 genes with base substitution were inserted were prepared and introduced into Escherichia coli to express R-020 variants. The resulting variants were purified and their enzyme inhibitory activities were measured. The FXa-inhibitory activity was increased in four variants with single amino acid substitution. With another four variants having two amino acid substitutions involving combinations of the above single amino acid substitutions, the FXa-inhibitory activity was further increased. Because the electrostatic interaction within R-020-FXa complex seemed stronger in these R-020 variants, this increase in FXa-inhibitory effect was speculated to be a consequence of more potent binding between the enzyme and the inhibitor.
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  • Kyoko Kohno, Kayoko Yasuzawa, Mika Hirose, Yasunobu Kano, Naoki Goshim ...
    1994 Volume 115 Issue 6 Pages 1113-1118
    Published: 1994
    Released: November 18, 2008
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    The molecular mechanism of autoregulation of expression of the hupA gene in Escherichia coli was examined. The promoter of the gene contains a palindromic sequence with the potential to form a cruciform DNA structure in which the -35 sequence lies at the base of the stem and the -10 sequence forms a single-stranded loop. An artificial promoter lacking the palindrome, which was constructed by replacing a 10 nucleotide repeat for the predicted cruciform arm by a sequence in the opposite orientation, was not subject to HU-repression. DNA relaxation induced by deleting HU proteins and/or inhibiting DNA gyrase in cells results in increased expression from the hupA promoter. We propose that initiation of transcription of the hupA gene is negatively regulated by steric hindrance of the functional promoter domains for formation of the cruciform configuration, which is facilitated at least in part by negative supercoiling of the hupA promoter DNA region. The promoter region of the hupB gene also contains a palindromic sequence that can assume a cruciform configuration. Negative regulation of this gene by HU proteins may occur by a mechanism similar to that operating for the hupA gene.
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  • Hironao Ueno, Takashi Masuko, Jue Wang, Yoshiyuki Hashimoto
    1994 Volume 115 Issue 6 Pages 1119-1127
    Published: 1994
    Released: November 18, 2008
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    Antigenic determinants (epitopes) on bovine serum albumin (BSA) to anti-BSA monoclonal antibodies (mAbs) were assessed by means of photoaffinity labeling. Anti-BSA mAbs, AB3 and AB6, were modified with an 125I-labeled photoreactive crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1, 3'-dithiopropionate (SASD) and then bound with BSA. The complex yielded was irradiated with UV light to facilitate covalent binding of the modified mAb with BSA. Reduction of the product with 2-mercaptoethanol revealed that radioactivity of 125I-SASD was specifically introduced into BSA, probably near the mAb combining site (s). The radiolabeled or native BSA was digested with V8 protease and the peptides produced were assessed for their amino acid sequence, the radiolabeled amino acids contained, and their reactivity to mAb. The results indicated that AB3 mAb recognized an epitope in the region of Ala-537 to Asp-554 on a linear part of the BSA molecule, whereas AB6 mAb probably recognized an epitope of the assembled form present in a peptide of Asp-299 to Glu-338.
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  • Miyuki Shimoji, Yoko Aniya
    1994 Volume 115 Issue 6 Pages 1128-1134
    Published: 1994
    Released: November 18, 2008
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    Glutathione S-transferases in testis microsomes were purified from rats and compared with the liver microsomal transferase. When microsomal fractions were prepared from rat testis by the same method as used for liver microsomes, testis microsomal glutathione S-transferase activity was increased 2-fold by N-ethylmaleimide as compared to a 7-fold increase in that of the liver transferase. In contrast to the single glutathione S-transferase in liver microsomes, at least three isozymes of glutathione S-transferase were separated from testis microsomes on hydroxylapatite column chromatography. The major fraction exhibiting glutathione S-transferase activity from the testis microsomes was shown to contain a member of the Mu family. The second fraction with transferase activity contained one of the Alpha class, and the third and smallest fraction was found to contain the liver microsomal form of glutathione S-transferase. Since the GSH S-transferase of the Mu family is present in the cytosol, we isolated the GSH S-transferase from testis cytosol, it being suggested that the major GSH S-transferase in testis microsomes is the cytosolic transferase. These results indicate that testis microsomes contain mainly the cytosolic form of glutathione S-transferase, and that the activity of the liver microsomal form of the transferase is very low.
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  • Hiroko Hama, Takashi Kayahara, Wakano Ogawa, Masaaki Tsuda, Tomofusa T ...
    1994 Volume 115 Issue 6 Pages 1135-1140
    Published: 1994
    Released: November 18, 2008
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    When cells of Escherichia coli are grown on lactate (or other carbon sources), an addition of serine to the medium causes growth inhibition. This growth inhibition is caused by inhibition by serine of homoserine dehydrogenase I, which is involved in threonineisoleucine biosynthesis [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. We have cloned and sequenced genes which enhance the serine-sensitivity. Two open reading frames were found and designated as sseA and sseB. Introduction of either sseA or sseB gene, or both, into E. coli cells enhanced the serine-sensitivity. The sseA gene elicited stronger enhancement than sseB. The deduced amino acid sequence of SseA showed considerable similarity with that of bovine liver rhodanese, which catalyzes sulfur transfer from thiosulfate. We observed a twofold increase in rhodanese activity in E. coli cells harboring a plasmid carrying the sseA gene. The position of sseA in the genetic map is around 52'. However, sseA is different from cysM, which codes for O-acetylserine sulfhydrylase-B, an enzyme catalyzing sulfur transfer from thiosulfate to O-acetylserine, the map position of which is also around 52'.
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  • Masao Nakamura, Takaaki Hayashi
    1994 Volume 115 Issue 6 Pages 1141-1147
    Published: 1994
    Released: November 18, 2008
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    NAD (P) H-quinone (menadione, Trolox C quinone, and α-tocopherol quinone) reductase activity of rat liver subeellular fractions was observed optically at 340-400 nm, and oxygen radical generation was demonstrated using the ESR spin trap, 5, 5'-dimethyl-l-pyrroline-1-oxide. NAD (P) H-menadione reductase activity of the fractions decreased in the order: cytosol>microsomes>plasma membranes. Although more than 65% of the activity of microsomes and plasma membranes was inhibited on the addition of dicoumarol, no change in the menadione-mediated formation of oxygen radicals by either fraction was observed. As judged from the intensity of ESR signals, the menadione-mediated oxygen radical formation by plasma membranes was only one-tenth as great as that by microsomes. No generation of oxygen radicals in the NAD (P) H-menadione reductase reaction by cytosol was found, and the activity was abolished in the presence of dicoumarol, an inhibitor of DT-diaphorase. It is concluded that plasma membranes reduce quinones by way of two-electron transfer and that the activity may prevent cellular quinone toxicity. NAD (P) H-α-tocopherol quinone reductase activity was confirmed in all cellular fractions [Hayashi et al. (1992) Biochem. Pharmacol. 44, 489-493] and this activity was also inhibited by dicoumarol, suggesting that it was due to DT-diaphorase.
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  • Hiroshi Kurata, Takayoshi Sannoh, Yasunori Kozutsumi, Yasunori Yokota, ...
    1994 Volume 115 Issue 6 Pages 1148-1154
    Published: 1994
    Released: November 18, 2008
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    Mannan-binding proteins (MBPs) occur in two forms, both of which are synthesized in the liver. Although two different MBP cDNAs have been cloned and characterized for rat and mouse, only one form of human MBP eDNA has been isolated. In this study, two forms of human MBP, liver MBP (L-MBP) and serum MBP (S-MBP), were purified from liver and serum and characterized, respectively. The amino acid sequences of these two human MBPs were identical and consistent with those deduced from the cDNA sequence. The most significant difference between L-MBP and S-MBP was the number of subunits, which was about 9 in L-MBP and 18 in S-MBP. Furthermore, S-MBP but not L-MBP had the ability to activate the complement. These results suggested that a newly synthesized protein is processed post-translationally into two forms, S-MBP and L-MBP, in human liver. Recombinant MBP synthesized in COS-1 cells, after transfection with human MBP eDNA, was secreted into the medium, suggesting that COS-1 cells lack a mechanism for differentiating S-MBP and L-MBP. A mutant MBP synthesized in COS-1 cells which lacked a sequence comprising 9 amino acid residues at the beginning of the collagen-like domain had no ability to activate the complement, suggesting that this sequence plays an important role in the activation of the complement by human MBP.
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  • Hiroyuki Mizuguchi, Seiji Ito, Valery I. Shevchenko, Yasuyuki Nagasawa ...
    1994 Volume 115 Issue 6 Pages 1155-1161
    Published: 1994
    Released: November 18, 2008
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    Rat C6 astroglioma cells (C6-bH1R cells) expressing cloned bovine histamine H1 receptors were established by transfection with a vector (pEF-BOS-bH1R) which carried a 2.7-kbp EcoRI fragment of the bovine H1 receptor cDNA [Yamashita, M. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 11515-11519]. The cloned bovine H1 receptor in C6-bH1R cells was characterized by three established criteria: the [3H]mepyramine binding assay, the accumulation of inositol phosphates induced by histamine, and histamine-induced elevation of intracellular Ca2+ concentration, ([Ca2+]1). The accumulation of inositol phosphates induced by histamine was time- and dose-dependent. The accumulation of inositol trisphosphate was biphasic with a prompt increase to the maximal level, followed by a sustained submaximal level. The histamine-induced accumulation of inositol phosphates was suppressed by phorbol ester, but not by pertussis toxin. Results from the [3H]-mepyramine binding assay and histamine-induced elevation of [Ca2+]1 were characteristic of H1 receptors. Several compounds among tricyclic antidepressants, neuroleptics, and serotonin antagonists showed affinities to the cloned bovine H1 receptor with K1 values similar to reported values. Histamine neither induced cAMP accumulation nor attenuated forskolin-induced cAMP accumulation in C6-bH1R cells. C6-bH1R cells are particularly useful for studying the H1 receptor-mediated astroglial cell functions.
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  • Teruo Kuroda, Toshi Shimamoto, Kei Inaba, Takashi Kayahara, Masaaki Ts ...
    1994 Volume 115 Issue 6 Pages 1162-1165
    Published: 1994
    Released: November 18, 2008
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    The properties of the Na+/H+ antiporter in Vibrio parahaemolyticus, a slightly halophilic bacterium, were investigated using everted membrane vesicles. It appears that at least two Na+/H+ antiporters are present, one that is pH-dependent and one that is pH-independent. These two antiporters appear to correspond to the NhaA and NhaB antiporters of Escherichia coli, respectively. It seems that amiloride strongly inhibits the pH-dependent antiporter. Na+ is the best substrate for both of the two V. parahaemolyticus antiporters. Li+ is a poorer substrate and K+ is not a substrate. No K+/H+ antiport activity was detected in membrane vesicles of this organism. The Na+(Li+)/H+ antiport activity greatly increased with an increase in pH of the assay medium. pH did not affect the Km value of the Na+/H+ antiport, but it did increase the Vmax.
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  • Toshimasa Yasuhara, Yasuko Mera, Toshiki Nakai, Akira Ohashi
    1994 Volume 115 Issue 6 Pages 1166-1171
    Published: 1994
    Released: November 18, 2008
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    An ATP-dependent proteolysis system in yeast mitochondria was characterized by examining the hydrolysis of mitochondrial translation products in isolated mitochondria. Degradation of [35S] methionine-labeled polypeptides synthesized in isolated yeast mitochondria was activated by exogenously added ATP. ADP, GTP, and CTP substituted for ATP to some extent, but nonhydrolyzable ATP analogues did not. Adenosine-5'-O-(3'-thiotriphosphate) effectively competed with ATP as activator. Carboxyatractyloside, an inhibitor of adenine nucleotide translocation across the mitochondria) inner membrane, and the metal chelator o-phenanthroline inhibited the ATP-dependent proteolysis. The latter inhibition was abolished by subsequent addition of Mn2+ or Co2+ but not Ca2+ or Zn2+. Hemin inhibited the ATP-dependent proteolysis of mitochondrial translation products with a half-maximum inhibition at 12 μM. Analysis by SDS-polyacrylamide gel electrophoresis showed that [35S] methionine-labeled polypeptides were rapidly degraded into low-molec-ular-weight species. Submitochondrial particles retained the ATP-dependent proteolytic activity and had the same spectrum of inhibitors as intact mitochondria except for a reduced effect of carboxyatractyloside. These results indicate that yeast mitochondria contain an ATP-dependent and hemin-sensitive proteolysis system which is associated with the inner membrane and can hydrolyze mitochondrial translation products, and that a chelator-sensitive protease is probably involved in this system.
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  • Takahiro Kusakabe, Kiyohisa Motoki, Katsuji Hori
    1994 Volume 115 Issue 6 Pages 1172-1177
    Published: 1994
    Released: November 18, 2008
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    To study the structure/function relationship and enzymatic properties of human aldolase C, we have constructed an Escherichia coli expression plasmid, pHAC11, for the isozyme. E. coli cells carrying this plasmid produced enzymatically active human aldolase C. The kcat and Km values for fructose-1, 6-bisphosphate (Fru-1, 6-P2) and fructose-1-phosphate (Fru-1-P) of the recombinant enzyme were found to be similar to those of authentic aldolase C from human brain. The Fru-1, 6-P2/Fru-1-P activity ratio of the recombinant enzyme is approximately 13.5, which is comparable to that of the recombinant rat aldolase C, but is slightly higher than those of rat brain and hepatoma aldolases C. The substitution of Ser for the carboxyl-terminal Tyr (Tyr-363) of the recombinant enzyme caused a marked decrease in that of Fru-1, 6-P2, with little change in that of Fru-1-P. The activity ratio changed from 13.5 for the normal enzyme to 3.8 for the engineered enzyme. Human aldolase C was found to form tetrameric hybrids with aldolase B in vivo when these enzymes were coexpressed in E. coli cells.
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  • Shunsuke Nagao, Takaomi C. Saido, Yoshiko Akita, Takahide Tsuchiya, Ko ...
    1994 Volume 115 Issue 6 Pages 1178-1184
    Published: 1994
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    We examined the activation of μ-calpain in human epidermoid carcinoma KB cells following a rise in intracellular calcium concentration using antibodies specifically recognizing different activation states of μ-calpain. KB cells possess calpastatin activity in large excess of calpain activity as analyzed by ion exchange HPLC. Stimulation of the cells with a calcium ionophore, ionomycin, caused production of the autolytic intermediate form (Mr=78 k) of μ-calpain derived from the preautolysis form (80 k), while the fully autolyzed postautolysis form (76 k) remained below detectable levels at all times. The appearance of the autolytic intermediate paralleled limited proteolysis of the membrane-associated calpastatin fractions (110 k and 106 k); the resulting fragments (68 k and 45 k) were released into the cytosol. Both the production of the autolytic μ-calpain intermediate and the limited proteolysis of calpastatin in cell lysates in the presence of calcium were inhibited by a synthetic calpastatin peptide, indicating that proteolysis of calpastatin was indeed catalyzed by calpain and that the autolytic intermediate may have exerted the proteolytic activity. Furthermore, μ-calpain autolysis and calpastatin degradation, upon ionomycin treatment, were both augmented by epidermal growth factor (EGF). These results suggest that calpastatin serves not only as an inhibitor but also as a substrate for calpain at cell membranes and that intracellular conditions associated with the cell cycle may affect the activation of μ-calpain.
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  • Tadashi Shimamoto, Kazuo Noguchi, Masayuki Kuroda, Masaaki Tsuda, Tomo ...
    1994 Volume 115 Issue 6 Pages 1185-1189
    Published: 1994
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The organization of the melibiose operon of Escherichia coli is promoter-melA-melB. The amount of the product (α-galactosidase) of the first gene (melA) is much larger than that of the product (melibiose permease) of the second gene (melB). Using the chloramphenicol acetyltransferase gene (cat) as reporter, we found that there was an element between melA and melB, which reduced the expression of the downstream gene, melB. This region contained a boxA-like sequence, which is known as a binding site for an attenuation factor, NusA. Northern hybridization analysis revealed that the ratio of melA mRNA and melAB mRNA was comparable with the ratio of the melA and melB products. We also found thai the melA mRNA was about 3-fold more stable than the melAB mRNA. Experimental results obtained with a nusAts mutant suggested that the NusA protein is involved in the reduced expression of the melB gene. We conclude that the production ratio of α-galactosidase and melibiose permease is regulated at two levels: 1) transcription and 2) mRNA stability.
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  • Hideyuki Komatsu, Katsuhisa Tawada
    1994 Volume 115 Issue 6 Pages 1190-1196
    Published: 1994
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Lys-83 in the heavy chain of rabbit skeletal muscle myosin is rapidly and stoichiometrically modified by trinitrobenzene sulfonate. Other authors claimed that the half-stoichio-metric trinitrophenylation of Lys-83 in myosin in the presence of PPi, was correlated to a Pro/Ser microheterogeneity at the 78 th residue position in the heavy chain [Miyanishi, T., Maita, T., Matsuda, G., & Tonomura, Y. (1982) J. Biochem. 91, 1845-1853]. However, our recent studies with chymotryptic subfragment 1 (S1) instead of myosin showed no such correlation between the half-stoichiometric trinitrophenylation and the Pro/Ser micro-heterogeneity [Komatsu, H. & Tawada, K. (1993) J. Biol. Chem. 268, 16974-16978]. Since the global structure of the head portion of myosin is different from that of chymotryptic S1 that lacks DTNB light chain, it could be argued that the difference is due to the structural difference between chymotryptic S1 and myosin. We hence reexamined the situation with myosin, and obtained the same results as found with S1: (i) Lys-83 in myosin was half-stoichiometrically trinitrophenylated in the presence of PPi, although it was stoichiometrically modified in the absence of PPi; (ii) there was a Pro/Ser microheterogeneity at the 78 th position in the myosin heavy chain, which was not correlated to the half-stoichiometric trinitrophenylation of Lys-83 in the presence of PR.
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  • Isao Ishii, Michio Ui
    1994 Volume 115 Issue 6 Pages 1197-1202
    Published: 1994
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Culture of mouse resident peritoneal macrophages with retinoic acid resulted in increased expression of the tissue transglutaminase gene as revealed by increases in the maximal velocity of the enzyme reaction in the cytosol and in the enzyme mRNA level. Protein kinase C-activating phorbol esters and okadaic acid, both of which were without effect on the enzyme induction by themselves, enhanced the retinoic acid-induced gene expression, which was in turn inhibited partially by pertussis toxin and totally by inhibitors of protein kinase C in either the presence or absence of phorbol esters. Retinoic acid was more effective in the “conditioned” medium, in which macrophages had been cultured for a time longer than 4 h, than in the “fresh” medium. The retinoic acid induction of transglutaminase was accompanied by increased phosphatidylinositol turnover and phosphatidic acid generation, which were efficiently suppressed by prior exposure of cells to pertussis toxin. It is likely that certain autocrine factor (s) liberated during culture of macrophages may afford conditions favorable for retinoic acid-induced gene expression, presumably via pertussis toxin-sensitive G protein-mediated phosphoinositide metabolism leading to activation of protein kinase C.
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