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Volume 114 (1993)
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Volume 114, Issue 6
Displaying 1-30 of 30 articles from this issue
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Tsuyoshi Inoue, Nobuya Nishio, Yasushi Kai, Shigeharu Harada, Yoshiki ...1993 Volume 114 Issue 6 Pages 761-762
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNew crystals of a blue copper protein, pseudoazurin from denitrifier Achromobacter cycloclastes IAM 1013, have been obtained by means of vapor diffusion with ammonium sulfate as a precipitant at pH 6.0 and 4°C. The crystals belong to the orthorhombic system, space group P212121, with unit cell dimensions of α=56.69 (2), b=61.53 (2), and c=30.20 (1) Å. The asymmetric unit includes one molecule of pseudoazurin with a Vm value of 2.04 Å3/Da. The crystals are so stable against X-ray irradiation that a complete data set up to 1.54 Å has been collected using a single native crystal. Solution of the structure was performed by means of the Patterson search techniques, and the current crystallographic R-factor is 17.5% at 3.0 Å resolution. Refinement at higher resolution is in progress.View full abstractDownload PDF (1183K) -
Nahoki Kuraya, Kaoru Omichi, Hitoshi Nishimura, Sadaaki Iwanaga, Sumih ...1993 Volume 114 Issue 6 Pages 763-765
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSType and structural analysis of O-linked sugar chains in human blood clotting factor IX was performed by the pyridylamination method developed for O-linked sugar chains [Kuraya, N. & Hase, S. (1992) J. Biochem. 112, 122-126]. O- and N-linked sugar chains were released with hydrazine, and then N-acetylated, followed by pyridylamination. The type of sugar chain was determined by reducing-end analysis of the pyridylaminated (PA-) sugar chains. Sugar chains with PA-GalNAc at the reducing terminal and that with PA-Fuc [Nishimura, H. et al. (1992) J. Biol. Chem. 267, 17520-17525] were obtained besides known sugar chains with PA-Glc from the Xyl-Glc-Ser type and those with PA-GlcNAc from asparagine-linked sugar chains. The sugar chains with PA-GalNAc were identified as mono- and disialyl Galβ1-3GalNAc by two-dimensional HPLC mapping. The structure of the sugar chain with PA-Fuc was Neu5Acα2-6 Galβ1-4GlcNAcβ1-3 Fuc, as determined by exoglycosidase digestion, methylation analysis, and Smith degradation.View full abstractDownload PDF (295K) -
Katsuko Yamashita, Takashi Ohkura, Hiroko Ideo, Kousaku Ohno, Michiko ...1993 Volume 114 Issue 6 Pages 766-769
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that the carbohydrate-deficient glycoprotein (CDG) syndrome is an asparagine-N-linked sugar chain transfer deficiency [Yamashita et al. (1993) J. Biol. Chem. 268, 5783-5789]. In order to confirm this hypothesis, we applied electrospray ionization-mass spectrometric analysis to transferrin isoforms purified from patients with the CDG syndrome. Transferrin isoforms containing 4, 2, and 0 sialic acid residues, S4, S2, and S0, were separated by Mono Q anion exchange column chromatography from serum of a patient with the CDG syndrome. The molecular masses of S4•S2, and S0 were determined to be 79, 570±5, 77, 364±6, and 75, 157±6 Da by electrospray ionization mass spectrometry (ESI/MS). The differences between S4 and S2, and between S2 and S0 were both in accordance with the molecular mass of a disialylated biantennary sugar chain {Neu5Acα2→6Ga1β1→•G1cNAc β1→2Man α 1→6 (Neu5Ac α 2→6Gal β 1→4G1cNAc β 1→2Man α 1→3) Man β1→ 4G1cNAcβ1→4G1cNAc} (2, 206 Da), showing that S0 is nonglycosylated, and that S4 and S2 carry 2 and 1 mol of asparagine-N-linked sugar chains, respectively. The nonglycosylated asparagine site of S2 was elucidated to be random by high performance liquid chromatog-raphy-ESI/MS of a tryptic peptide of reduced and pyridylethylated S2. ESI/MS analysis of transferrin purified through one step from serum is applicable for a definite diagnosis of the CDG syndrome.View full abstractDownload PDF (345K) -
Keiichi Yamamoto1993 Volume 114 Issue 6 Pages 770-772
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe describe cross-linking conditions for myosin subfragment-1 and actin with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide, with which the compositions of cross-linked species can be altered. The specific Mg-ATPase activity of cross-linked subfragment-1 in these preparations was almost the same in spite of the differences in the compositions of the cross-linked species. This suggests that the two kinds of cross-linked species have almost the same ATPase activity, and that the cross-linking at these different sites nearly equally facilitates the formation of the strong binding state which accompanies the release of ADP and Pi.View full abstractDownload PDF (1280K) -
Takayoshi Iio1993 Volume 114 Issue 6 Pages 773-778
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+-dependent interaction between skeletal muscle troponin C and a bee venom melittin, which can be regarded as a mimic of the troponin C-binding peptide of troponin I, was investigated. Sephadex gel chromatography revealed that melittin bound to troponin C irrespective of the presence or absence of Ca2+ in 50mM KCl and 50mM Tris-HCl, pH 7.5. At high salt concentration, 0.5-1.2M KCl, melittin was removed from apo troponin C but still bound to Ca2+-loaded troponin C. Hydrophobic affinity chromatography revealed that hydrophobic region (s) appeared on the surface of troponin C upon Call binding but vanished upon Ca2+ release. Therefore, at physiological salt concentration, melittin binds to Ca2+-loaded troponin C by both hydrophobic and electrostatic interactions, and it binds to apo troponin C by electrostatic interaction. The midpoint of the fluorescence titration curve of troponin C-melittin complex, observed by Trp-19 fluorescence, scarcely depended on the concentration of MgCl2. This means Trp-19 of melittin detects only the confor-mational change of troponin C induced by Call-binding to the low affinity Ca2+-binding sites (sites I and II) of troponin C; that is, the C-terminal of melittin binds to the N-terminal of troponin C. Fluorescence stopped-flow experiments revealed that the time course of Trp-19 fluorescence change induced by the Ca2+-release from the low affinity Ca2+-binding sites of troponin C in the complex was biphasic. An analysis of the rate constants of the reactions revealed that the fast phase reflected the Ca2+-release reaction from the low affinity sites, and the slow phase reflected the resultant structural change of the complex.View full abstractDownload PDF (633K) -
Kiyoto Motojima, Akira Ohmori, Yasuko Takino, Sataro Goto1993 Volume 114 Issue 6 Pages 779-785
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe changes in protein phosphorylation during induction and deinduction of peroxisome proliferation in rat liver by three types of proliferators were studied by in vitro phospho-rylation assay. Among the variously phosphorylated proteins, an increase during induction and a decrease during deinduction in phosphorylation of P 100, a cytosolic protein having a molecular weight of 100 kDa, was most remarkable. The time course of enhance-ment of phosphorylation by the administration of the proliferators, however, was not parallel with proliferation of peroxisome but with increase in the liver DNA content. Amino acid sequencing of the protein indicated the identity of its N-terminal 17 amino acid residues with those of elongation factor 2 (EF 2). Increase in the amount of EF 2 by peroxisome proliferators was confirmed by immunoblotting and this was almost parallel with peroxisome proliferation, suggesting that both increase in the amount of EF 2 and some changes in phosphorylation activities account for a large increase in in vitro phosphorylation of EF 2 by the administration of peroxisome proliferators.View full abstractDownload PDF (3977K) -
Seema Qamar, Mozaffarul Islam, Saad Tayyab1993 Volume 114 Issue 6 Pages 786-792
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChemical modification was used as a probe to study the effect of structural features of serum albumin (charge, conformation, surface hydrophobicity, etc.) on its solubility behavior in concentrated ammonium sulfate solutions. Four different acetylated derivatives of goat serum albumin namely 18% acetylated, 40% acetylated, 53% acetylated, and 93% acetylated albumins were prepared. The homogeneity of these preparations was established by gel chromatography and polyacrylamide gel electrophoresis. Hydrodynamic data on the Stokes radius of native and acetylated albumins suggested gradual change in conformation on increasing modification. Solubility experiments performed in concentrated ammonium sulfate solutions at pH 7.0 and at 30°C showed a slight decrease in salting-out parameter, Ks, up to 40% modification, whereas a significant decrease was obtained at higher modification. However, the salting-out parameter, β, decreased monotonously. Similar decrease in these parameters was also observed with different modified albumins at other pH values viz. pH 5.5, 4.5, and 3.6. From these results we conclude that the decrease in solubility of serum albumin on increasing modification was primarily due to change in conformation.View full abstractDownload PDF (1260K) -
Fumio Watanabe, Yoshiyuki Tamura, Erhard Stupperich, Yoshihisa Nakano1993 Volume 114 Issue 6 Pages 793-799
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCobalamin uptake by Euglena mitochondria is a biphasic process, consisting of energy-independent cobalamin-binding to mitochondrial membranes and energy-dependent active transport. The energy-dependent phase of cobalamin uptake is not dependent on mitochondrial respiration, but on the presence of ATP within the mitochondrial matrix. The dissociation constant of the energy-independent cobalamin-binding reaction is estimated to be 0.45 nM. Inhibition of the mitochondrial cobalamin uptake by a variety of cobalamin analogues indicates that Euglena mitochondria have an absolute requirement for the complete cobalamin molecule with an α-axial ligand (the cobalt-coordinated nucleotide) and an intact b-propionamide side-chain. Thus, the Euglena mitochondrial cobalamin uptake system is highly specific for the cobalamin structure. The cobalamin taken up by the Euglena mitochondria cannot be exchanged with exogenous cobalamin. All of the mitochondrial cobalamin is associated with three proteins with molecular masses of>700, 000 (16.3%), 160, 000 (7.4%), and 35, 000 (76.3%). They occur in the soluble fraction of mitochondria, suggesting that these cobalamin-binding proteins or cobalamin-dependent enzymes play an important role in cobalamin accumulation and metabolism within the mitochondria.View full abstractDownload PDF (725K) -
Hideaki Watanabe, Hiroaki Narumi, Tomio Inaba, Kazuko Ohgi, Masachika ...1993 Volume 114 Issue 6 Pages 800-807
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA ribonuclease (RNase Oy) was purified to homogeneity on SDS-PAGE from the homogenate of oyster (Crussdstrea grigus). The apparent molecular weight estimated from SDS-PAGE was ca. 28 kDa. The pH optimum of the RNase was 5.0. The RNase released mononucleotides from RNA in the order of 3'-GMP, 3'-AMP, and 3'-UMP. The complete amino acid sequence of RNase Oy was determined, mostly by analyzing the peptides generated by BrCN cleavage or digestion by lysylendopeptidase, staphylococcal V8 protease, and α-chymotrypsin. The molecular weight of the protein moiety of RNase Oy deduced from the sequence was 24, 359. The sequence of RNase Oy contained two typical histidine residues in segments common to the active site of RNase T2 family enzymes. The locations of six half cystine residues among eight were almost superimposable on those of four known plant RNases of RNase T2 family. The sequence homology between RNase Oy and five fungal and four plant RNases amount, to 43-56 amino acid residues. The amino acid sequence of the N-terminal part of RNase Oy is more similar to those of plant RNases than to those of fungal RNases. This RNase is the first RNase T2 family RNase from mollusc whose primary structure has been eluci-dated.View full abstractDownload PDF (1522K) -
Torn Hisabori, Katsura Mochizuki1993 Volume 114 Issue 6 Pages 808-812
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inactivation and the activation of the ATPase of isolated CF1 as assayed by the hydrolysis of ATP in 10 s depended on prior binding of ADP-Mg and ATP-Mg. The effects of Mg2+ on the nucleotide binding kinetics were studied by monitoring the time courses of UV spectral changes induced by the interaction between CF, and ADP or ATP using a rapid-scan spectrophotometer equipped with a stopped-flow cell. The apparent rate constant of ADP binding to the two high-affinity sites on CF, (designated sites B and C in the previous report [Hisabori, T. & Sakurai, H. (1984) Plant Cell Physiol. 25, 483-493]) was drastically increased by prior binding of Mg2+ to CF1 but not ATP. The inhibitory effect of Mg2+ was attributed to a marked increase in kon for the inhibitory ADP binding at the high-affinity sites induced by the previous binding of Mg2+ to the enzyme. The location of site B is suggested to be on the β subunit based on the difference spectral change induced by binding of the ADP analog 2', 3'-O-(2, 4, 6-trinitrophenyl) ADP to CF1.View full abstractDownload PDF (547K) -
Fumitaka Oyama, Kimikazu Hashino, Rieko Oyama, Ikunoshin Kato1993 Volume 114 Issue 6 Pages 813-819
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSKunitz-type serine proteinase inhibitor (KPI) domain of Alzheimer's disease-related β-amyloid protein precursor (APP) was expressed in Escherichia coli as a fusion protein with a truncated form of Staphylococcus protein A. The fusion protein was purified from the cell culture medium using an IgG Sepharose column. The KPI domain was separated from the protein A portion by cleavage with human α-thrombin at the engineered recognition sequence, followed by purification on IgG Sepharose and reversed-phase HPLC columns. The recombinant KPI domain strongly inhibited trypsin; the inhibition constant (Ki) for bovine trypsin was 2.5×10-11M, comparable to those of the secreted forms of APP with the KPI domain. The recombinant protein contained three intramolecular disulfide bonds, which were determined to be located between Cys-6 (C 1) and Cys-56 (C 6), Cys-15 (C 2) and Cys-39 (C 4), and Cys-31 (C 3) and Cys-52 (C 5) of the recombinant KPI domain, respectively. These positions are highly homologous to those of disulfide bonds in bovine pancreatic trypsin inhibitor. The trypsin-inhibitory activity of the recombinant protein was abolished by preincubation with 0.4mM dithiothreitol under non-denaturing conditions. By this mild reduction, all the disulfide bonds were completely cleaved. These results clearly indicate that the disulfide bonds play an important role in the function of the KPI domain of APP.View full abstractDownload PDF (764K) -
Kunio Matsumoto, Hisao Tajima, Hiroko Okazaki, Toshikazu Nakamura1993 Volume 114 Issue 6 Pages 820-826
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHepatocyte growth factor (HGF) has mitogenic and unique morphoregulatory functions, and is considered to act as a hepatotrophic and a renotrophic factor for regeneration of the liver and kidney subjected to various insults. We have now obtained evidence that heparin is a potent inducer of HGF production. The addition of heparin to a culture of MRC-5 human embryonic lung fibroblasts increased the HGF concentration in the conditioned medium, in a dose-dependent manner. The maximal stimulation was obtained at 1μg/ml heparin and stimulation was 4-fold compared to control cultures. The rate of HGF synthesis in MRC-5 cells, as measured by pulse-labeling with [35S] methionine, and subsequent immuno-precipitation of HGF from both conditioned medium and a cell lysate, was 3-4-fold stimulated by 1μg/ml heparin, whereas heparin apparently had no significant effect on the HGF mRNA level. The stimulatory effect of heparin on HGF production was evident in various types of cells, such as MRC-9, IMR-90, and WI-38 human embryonic lung fibro-blasts, human skin fibroblasts, HL-60 human promyelocytic leukemic cells and human umbilical vein endothelial cells. In addition to heparin, heparan sulfate also stimulated HGF production, albeit to a lesser extent than heparin; 1.7-fold stimulation with 2μg/ml heparan sulfate. However, other glycosaminoglycans, such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, and keratan polysulfate, had no stimulatory effect on HGF production. Interleukin-lα (IL-1α) and 12-O-tetradecanoylphorbol-13-ace-tate (TPA) stimulated HGF production in human skin fibroblasts, and the simultaneous addition of heparin with IL-1α or TPA caused further marked enhancement of HGF production, in a synergistic manner. It would thus appear that heparin act as a potent inducer of HGF production by influencing post-transcriptional processes. We propose that heparin plays an important physiological role as an enhancer for tissue regeneration through its unique potential to stimulate HGF production.View full abstractDownload PDF (2311K) -
Toshihiro Fujii, Masatomo Watanabe, Yoshiro Ogoma, Yoshiyuki Kondo, Ta ...1993 Volume 114 Issue 6 Pages 827-829
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMicrotubule-associated protein (MAP)1 consisting of MAP 1 A and 1 B was purified from rat brain by the poly-L-aspartic acid (PLAA) method. We found that MAP 1 bound to F-actin in vitro up to a molar ratio of MAP 1 to actin monomers of 1:10. The apparent binding constant was about 2.7×107M-1. In contrast to the binding of MAP 2 or tau to F-actin, the binding of MAP 1 to F-actin did not affect the low-shear viscosity of actin filaments. Binding experiments performed using fragments of MAP 1, obtained by chymotrypsin digestion, indicated that MAP 1 included binding domains to F-actin that were different from those in microtubules and also two light chains (31 and 29 kDa) that were cosedimented with F-actin as well as with microtubules.View full abstractDownload PDF (985K) -
Kaoru Nagai, Haruki Yamaguchi1993 Volume 114 Issue 6 Pages 830-834
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was previously reported [Nagai, K. & Yamaguchi, H. (1993) J. Biochem. 113, 123-125] that intramolecular high-mannose chains are essential for reconstitution of soybean lectin from denatured subunits. To obtain more detailed information on the role of the intra-molecular high-mannose chains in the folding and assembly of soybean lectin polypep-tides, the effects of asparagine-linked oligosaccharides, Man9GIcNAc2Asn (M9-Asn) and Glc1-3Man9GlcNAc2Asn (GM9-Asn), on the reconstitution of soybean lectin from denatured subunits were examined by comparison with the denaturation features of the lectin with varying concentrations of guanidine hydrochloride. The combined use of spectroscopy and size-analysis by gel filtration revealed that both the folding and assembly of denatured subunit polypeptides were completely prevented in the presence of 300μM M9-Asn, whereas the same concentration of GM9-Asn only interfered with the polypeptide assembly, exhibiting no significant effect on the polypeptide folding. These results, considered together with those in the previous report, indicate that the sugar branch Manα1-2Man-α1-2Man linked to the 3 position of the β-mannosyl residue of the high-mannose chains functions in the folding of the subunit polypeptides, and that other branches participate in the subunit assembly.View full abstractDownload PDF (500K) -
Kaeko Kamei-Hayashi, Reiko Oshino, Saburo Hara1993 Volume 114 Issue 6 Pages 835-841
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of glutathione S-transferase α from guinea pig liver was determined. Glutathione S-transferase α was composed of two identical subunits, each comprising 218 amino acid residues. The amino acid sequence of glutathione S-transferase α exhibited 73% homology with that of human glutathione S-transferase Ha, 69% with that of rat glutathione S-transferase Ya, and 68% with that of rat glutathione S-transferase Yc, which are known to belong to class Alpha. From the above result, together with previous observations on its substrate specificity, it was concluded that glutathione S-transferase α belonged to class Alpha.View full abstractDownload PDF (605K) -
Keigo Gohda, Takeshi Itoh, Yasuyuki Hiramatsu, Ken-ichi Tomita, Satosh ...1993 Volume 114 Issue 6 Pages 842-848
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously reported the crystallization of a mutant RNase T1 (Y45W) with a synthetic modified trinucleotide ApGflpA [Hakoshima, T. et al. (1990) J. Biochem. 108, 695-698]. In the present report, we describe the crystal structure refined at 2.4 Å resolution. During the refinement process, we found that the ApGflpA molecule was cleaved at the phosphodiester bond between the 5'-terminal adenosine and the subsequent 2'-fluoroguano-sine. At the end of the refinement (R=17.1%), it was supposed that the resulting molecules, i.e., 3'AMP and GflpA, were separately bound to the enzyme. In the complex structure, the binding-site of the enzyme was occupied by the guanine base of GflpA via a similar interaction to that of the enzyme complexed with 2'GMP, while the phosphate group of GpA was not bound to the active site. The guanosine adopted the anti orientation on the glycosyl torsion angle with a C 2'-endo-C 3'-exo sugar pucker. This conformation resulted in the phosphate group protruding from the active site. The phosphate group of 3'AMP was bound to the active site of the enzyme and oriented itself toward the solvent region. This orientation was different from that of TAMP bound to the RNase T1 (Y45W).View full abstractDownload PDF (662K) -
Yuko Watanabe, Kiyoshi Kawakami, Yasutaka Hirayama, Kei Nagano1993 Volume 114 Issue 6 Pages 849-855
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA clone encoding a zinc finger protein (AREB6) was isolated from a HeLa cell expression library using a positive regulatory element (-102 to -58) of rat Na, K-ATPase α1 subunit gene (Atplal) as a probe. The clone is apparently an extended one of Nil-2-a originally isolated as a negative regulator of interleukin 2 gene [Williams, T. M. et al. (1991) Science 254, 1791-1794]. The open reading frame encodes 1, 124 amino acids. It contains 7 zinc-finger motifs arranged in two widely separated clusters. A glutamic acid-rich region is observed at the C terminus from residues 989 to 1123. Co-transfection of the AREB6 cDNA with Atplal fused to a reporter luciferase gene indicated that the AREB6 protein enhances or represses the promoter activity of the gene depending on the quantity of cDNA and on the cell type. The mRNA of AREB6 is expressed in heart and skeletal muscle, but not in liver, spleen, or pancreas. Genomic Southern analysis indicated that the gene encoding AREB6 is present as only one copy or two at most. Another cDNA clone obtained by using the same probe was identified as HEB [Hu, J. S., Olson, E. N., & Kingston, R. E. (1992) Mol. Cell. Biol. 12, 1031-1042]. Co-transfection of the cDNA enhanced or repressed the promoter activity of Atplal depending on the cell type. The binding regions of AREB6 and HEB are distinct from those of C1, C2, and C3 that were identified as binding complexes to ARE by gel retardation analysis using MDCK and B 103 cell nuclear extracts [Suzuki-Yagawa, Y., Kawakami, K., & Nagano, K. (1992) Mol. Cell. Biol. 12, 4046-4055].View full abstractDownload PDF (3190K) -
Atsuko Nagai, Azadeh Kheirolomoon, Daisaku Ohta1993 Volume 114 Issue 6 Pages 856-861
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHistidinol dehydrogenase (HDH) catalyzes two sequential oxidation reactions to produce histidine from histidinol via histidinaldehyde. In HDH proteins so far reported, two Cys residues are conserved. From the results of the studies on Salmonella typhimurium HDH, it has been proposed that one of these two conserved Cys residues is involved in the thiohemiacetal formation at the aldehyde oxidation step [Grubmeyer and Gray (1986) Biochemistry 25, 4778-4784]. To clarify the reaction mechanism, we investigated the role of the conserved Cys residues by site-directed mutagenesis in cabbage HDH. Thus, Cys-112, that corresponds to the catalytic Cys residue of the Salmonella enzyme, and the other conserved one, Cys-149, were replaced with Ala, Ser, or Phe. All the Cys-112 mutant HDHs catalyzed both the alcohol dehydrogenase and aldehyde dehydrogenase reactions, producing 1 mot of L-histidine during the reduction of 2 mol of NAD+, as did the wild type HDH. Site-directed mutagenesis at Cys-149 did not cause significant changes in the catalytic properties, either. These observations, together with the results of detailed comparison of the catalytic properties of mutant HDHs, clearly indicate that neither Cys-112 nor Cys-149 is involved in the reaction, and ruled out the involvement of thiohemiacetal formation in the histidinol dehydrogenase reaction.View full abstractDownload PDF (1247K) -
Naoki Itano, Kayoko Oguri, Hayao Nakanishi, Minoru Okayama1993 Volume 114 Issue 6 Pages 862-873
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Lewis lung carcinoma-derived low metastatic clone, P 29, with a capacity to induce a fibrotic stromal response of host tissue, exhibits tumorigenesis depending on an interstitial matrix formed by the induced stromal cells. Using this clone, in the present study we isolated and characterized a membrane-intercalated proteoglycan that mediates interaction between the tumor cells and interstitial matrix. The tumor cells were cultured in the presence of [3H] glucosamine and [35S] sulfate or [35S] methionine, and hydrophobic proteo-glycans were isolated by chromatography on DEAE-Sephacel and then Octyl-Sepharose CL-4 B. Proteoglycans with high affinity to the octylresidue were obtained from the cell layer but not to any significant extent from the medium. By CsCl density gradient centrifugation, they were separated into bottom, middle, and top subfractions, which were shown to consist of homogeneous species with estimated Mr values of 270, 000 (named CPGIIIB), 200, 000 (CPGIIIM), and 195, 000 (CPGIIIT), respectively, by gel filtration on Sepharose CL-4 B. These proteoglycans were intercalated into phosphatidylcholine liposomes, suggesting that they are all membrane-intercalated proteoglycans. Analyses of their glycosaminoglycans with chondroitinase ABC and heparitinase I plus II demonstrated that they all contain heparan sulfate as a major glycosaminoglycan (58-85%) and chondroitin 4-sulfate as a minor one (15-42%). Of these three proteoglycans, only CPGIIIB proteoglycan bound specifically to fibronectin-Sepharose 4 B under physiological conditions. Molecular analyses of this proteoglycan by Sepharose CL-4 B or SDS-PAGE before and after treatments with glycosaminoglycan degradation enzymes or trifluoromethanesulfonic acid demonstrated that CPGIIIB proteoglycan is a hybrid proteoglycan having heparan sulfate and chondroitin 4-sulfate chains on the same core protein with an Mr of 40, 000. Affinity chromatographies of the CPGIIIB proteoglycan on fibronectin-Sepharose 4 B after treatments with these enzymes demonstrated that it bound to fibronectin via its heparan sulfate chains. On the basis of the above results, we propose that the CPGIIIB proteoglycan mediates the interaction between the tumor cells and interstitial matrix.View full abstractDownload PDF (4273K) -
Chika Katagiri, Takeshi Kawamura, Shu-ichi Matsuzawa, Yusuke Mizuno, S ...1993 Volume 114 Issue 6 Pages 874-878
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe assay conditions for protein phosphatase 2 B (PP2B) in crude extracts from mouse lymphoid tissues and lymphocytes were extensively investigated. Under the conditions elucidated, the PP2B activity was measured in autoimmune-prone MRL/MpJ-lpr/lpr mice (MRL/1pr mice) and two control strains, MRL/MpJ- +/+ mice (MRL/+/+ mice) and C3H/HeJ mice. In the control mice, PP2B activity was distinctly higher in spleen and thymus than brain and liver. PP2B activity was further elevated in spleen of MRL/lpr mice than in the controls. Furthermore, we observed a specific increase in PP2B activity in T lympho-cytes from MRL/1pr mice as compared with in those from control mice. On the other hand, no alteration was observed in PP2B activity in B lymphocytes. These results suggest the involvement of PP2B in the abnormal signal transduction and proliferation of T lympho-cytes in MRL/1pr mice.View full abstractDownload PDF (1449K) -
Toshio Yamano, Kiyo Kuroda, Shigeru Fuji, Retsu Miura1993 Volume 114 Issue 6 Pages 879-884
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMolecular oxygen or cytochrome c has been described as the electron acceptor of the reaction of old yellow enzyme with NADPH. In this study, menadione was found to be a sensitive electron acceptor of the reaction under aerobic as well as anaerobic conditions. The Km, value of menadione for old yellow enzyme is as low as 2-3×10-7M in the presence or absence of superoxide dismutase. The rate enhancement of the cytochrome c reduction of old yellow enzyme with NADPH was about eight times in the presence of menadione. The rate increment was slightly higher under aerobic than anaerobic conditions. The rate enhancement by menadione enabled sensitive determination of the enzyme activity in the assay system, which contained NADPH, cytochrome c, menadione, and old yellow enzyme. In the reaction course, the semiquinone species of menadione was trapped by the reaction with t-butyl-α-phenylnitrone. The radical adduct was detected on EPR. The dyestuff, 2, 6-dichlorophenolindophenol, was found to be reduced ineffectively even in the presence of menadione; moreover, it was inhibitory in the NADPH consumption reaction. Methylene blue or Lauth's violet, known to be capable of semiquinone formation, also behaved, like menadione, as a mediator of electron transport to cytochrome c. On the basis of the experimental results, the occurrence of the one electron transfer of the old yellow enzyme reaction was emphasized.View full abstractDownload PDF (677K) -
Masahiro Iwakura, Tomokuni Kokubu, Shin-i-chi Ohashi, Shin-i-chi Ohash ...1993 Volume 114 Issue 6 Pages 885-889
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe “dihydrofolate reductase (DHFR) handle” [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45; Iwakura, M. & Tanaka, T. (1992) J. Biochem. 111, 638-642] fused with a penta-peptide, leucine enkephalin (LEK), has been applied in immunoassays for LEK and for the preparation of anti-LEK monoclonal antibody. DHFR fused with LEK (DHFR-LEK) was first utilized as an immobilized antigen in an enzyme-linked immunosorbent assay for LEK. By using a commercially available anti-LEK and peroxidase-linked anti-IgG, LEK could be quantified in the range between 0.1 ng/ml and 10μg/ml. By using a commercially available anti-LEK and the DHFR-LEK as an enzyme-labeled antigen, LEK was quantified in the range between 0.1 ng/ml and 1μg/ml by monitoring the recovery of the DHFR activity from the immuno-precipitates. By using the DHFR-LEK as an immunogen, three mouse monoclonal antibodies against LEK, but not DIIFR, were isolated. All three monoclonal antibodies were of IgG1 κ type. The large-scale preparation of two of these monoclonal antibodies, designated as anti-LEK-36 and anti-LEK-74, was carried out and their recognition specificities were studied by competitive binding assays. The IC50 values of LEK for the anti-LEK-36 and anti-LEK-74 were 3.74×10-6 and 4.6×610-6M, respectively. The competitive binding assays showed that recognition specificities of the two monoclonal antibodies were high and restricted to LEK and leucine-enkephalin (sulfated form). These results strongly suggest that the DHFR handle is useful in several immunological applications.View full abstractDownload PDF (532K) -
Takeshi Sato, Kiyoshi Furukawa, Dale E. Greenwalt, Akira Kobata1993 Volume 114 Issue 6 Pages 890-900
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN-Acetylgalactosamine is usually not a constitutive monosaccharide of Asn-linked sugar chains. Our previous study showed that the Asn-linked sugar chains of bovine CD 36 prepared from milk fat globule membranes (MFGM) contain this unique monosaccharide as the Ga1NAcβ1→4GlcNAc group [Nakata et al. (1993) Biochemistry 32, 4369-4383]. Western blot analysis of bovine MFGM glycoproteins with Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with either an α- or β-N-acetylgalactosamine residue, showed that WFA binding is observed for most of the protein bands as detected with Coomassie Brilliant Blue staining. However, no WFA binding was observed for protein bands after treatment of MFGM glycoproteins with N-glycanase. Structural analyses of the sugar chains released by hydrazinolysis from the MFGM glycoproteins by sequential exoglycosidase digestion and by methylation analysis revealed that oligosac-charides, which bound to a WFA-agarose column, are bi-, tri-, and tetraantennary complex-type and hybrid-type sugar chains with the GalNAcβ1→4GlcNAc group in their outer chain moieties, while oligosaccharides, which passed through the column, were of high-mannose-type, hybrid-type, and complex-type, of which the latter two groups contained the Galβ1→4GlcNAc groups. These results indicated that many bovine MFGM glycoproteins contain Asn-linked sugar chains with the GalNAcβ1→4GlcNAc group.View full abstractDownload PDF (2383K) -
Masao Hiraiwa, Toyoaki Yamauchi, Shoji Tsuji, Masatoyo Nishizawa, Tada ...1993 Volume 114 Issue 6 Pages 901-905
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acid sialidase [EC 3. 2. 1. 18], partially purified from human placenta by Con A-Sepha-rose adsorption and p-aminophenyl thio-β-D-galactoside-CH-Sepharose (PATG-Sepha-rose) affinity chromatographies, was activated by incubation at 37°C. This activation showed both time and temperature dependencies, with the most effective activation observed at 37°C in the pH range between 4.3 and 5.2. The influence of various protease inhibitors on its activation was investigated. Among the protease inhibitors tested, amastatin, an inhibitor of aminopeptidase A, significantly inhibited activation. The partially purified enzyme preparation contained aminopeptidase activity, which was inhibited by amastatin. Zinc ions inhibited either the activation of sialidase or the aminopeptidase activity in the enzyme preparation. These results suggest the possibility of participation of aminopeptidase function in the activation process of sialidase.View full abstractDownload PDF (492K) -
Keiko Masuda-Momma, Toyohiro Shimakawa, Kuniyo Inouye, Keitaro Hiromi, ...1993 Volume 114 Issue 6 Pages 906-911
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn ultraviolet absorption difference spectrum characteristic of the ionization change of a tyrosyl residue was observed on the binding of subtilisin BPN' with Streptomyces subtilisin inhibitor (SSI) at alkaline pH. This difference spectrum was considered to be induced by a pKa shift (from 9.7 to _??_ 11.5) of a tyrosyl residue of subtilisin BPN' in the interaction with carboxyls of SSI [Inouye et al. (1979) J. Biochem. 85, 1115-1126]. In the present paper, the tyrosyl residue in subtilisin BPN' and the carboxyls in SSI were identified by analyzing the difference spectrum using mutants of subtilisin BPN' and SSI: naturally occurring mutants and those prepared by site-directed and cassette mutagenesis. The difference spectrum disappeared on the binding of a mutant subtilisin BPN' of which Tyr104 was replaced by Phe (S-BPN'Y104F) and SSI at pH 9.8. The magnitude of the absorption difference was much smaller when subtilisin BPN' was bound with a mutant SSI of which both Glu67 and Asp68 were replaced by Gly than with the wild-type SSI. These lines of evidence indicated that the difference spectrum was caused by Tyr104 of subtilisin BPN' interacting with Glu67 and Asp68 of SSI. The binding of subtilisin BPN' and SSI is accompanied by an increase of tryptophan fluorescence, which is pH-dependent in the range of pH 7-11 [Uehara et al. (1978) J. Biochem. 84, 1195-1202]. In the present study, this pH-dependence of the fluorescence diminished when SSI bound with S-BPN'Y104F. This suggested that the fluorescence increase was due to Trp106 of subtilisin BPN' and was influenced by the ionization of Tyr104.View full abstractDownload PDF (659K) -
Kunihiko Tatsumi, Takeshi Yamauchi, Kunihiko Kiyono, Kazumasa Kishi, Y ...1993 Volume 114 Issue 6 Pages 912-918
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS3-Cyano-2, 6-dihydroxypyridine (CNDP) was identified as a potent inhibitor (IC50 value, 4.4 nM) of dihydrouracil dehydrogenase (DHUDase) [EC 1. 3. 1. 2], a rate-limiting enzyme in 5-fluorouracil (5-FU) degradation. The inhibitory activity of CNDP was about 2, 000 times that of uracil under our assay conditions. Kinetic analyses with partially purified enzyme from rat liver revealed that the mechanism of inhibition of DHUDase by CNDP was of mixed type with an inhibition constant (K) of 1.51 nM. CNDP had less effect on 5-FU phosphorylation than on 5-FU degradation. The inhibitory effect of CNDP on ribosylation of 5-FU was 600 to 1, 000 times less than that on DHUDase. Moreover, CNDP did not inhibit uridine kinase, thymidine kinase, or pyrimidine phosphoribosyltransferase. Coadministration of CNDP with 1-ethoxymethyl-5-fluorouracil (EM-FU) to rats with Yoshida sarcoma elevated the level of 5-FU in both the blood and the tumor and enhanced the antitumor effect of EM-FU. These findings indicated that CNDP would be a useful chemical modulator in chemotherapy with 5-FU or its prodrugs.View full abstractDownload PDF (813K) -
Masato Nakai, Toshiya Endo, Toshiharu Hase, Yoshikazu Tanaka, Bernard ...1993 Volume 114 Issue 6 Pages 919-925
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c1 subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c1 more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c1 to complement the respiration deficiency of yeast cells lacking only cytochrome c1 or both cytochrome c1 and the QCR6 protein. The mutant cytochrome c1 with the deletion of the first acidic region (Δ68-80) was still functional in the cytochrome c1-deficient strain. Mutant cytochrome c1 with the deletion of the second acidic region (Δ168-179) caused a decrease in the com-plementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant cytochrome c1 with altered charge distribution in the acidic regions (Asp170Asp171→Asn170Asn171 or Asp170Asp171→Asn170OLys171) made the cytochrome c1-deficient cells respiration-competent. On the other hand, mutant cytochrome c1 with the deletion of the first acidic region (Δ68-80) or altered charge distribution in the second region (Asp170Asp171→Asn170Lys171) did not restore the respiration deficiency of the cells lacking not only cytochrome c1 but also the QCR6 protein. These results indicate that the acidic regions in cytochrome c1 are essential for the ubiquinol-cytochrome c reductase activity in yeast cells in the absence of the QCR6 protein, and suggest the acidic regions of cytochrome c1 may promote binding of complex III to cytochrome c in cooperation with the QCR6 protein.View full abstractDownload PDF (1443K) -
Ryuichi Kato, Seiki Kuramitsu1993 Volume 114 Issue 6 Pages 926-929
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe recA gene of a thermophilic eubacterial strain, Thermus thermophilus (T. th.) HB 8, was cloned from a genomic DNA library by Southern hybridization using a gene-internal fragment amplified by the polymerase chain reaction (PCR) method as the probe. The gene encoded a 36 kDa polypeptide whose amino acid sequence showed 61% identity with that of the Escherichia coli RecA protein. Characteristic amino acid changes between the two RecA proteins were found. In the amino acid composition of the T. th. RecA protein, the number of Pro residues was increased, the number of Cys residues was decreased, and Lys residues were replaced by Arg, Asp by Glu, Thr by Val, and Ile by Val or Leu. These changes are supposed to stabilize the native protein conformation against heat denaturation. The amino acid residues in the nucleotide binding site of the protein and in the protein-protein interaction site responsible for the oligomer formation were well conserved. The T. th. recA gene has the ability to complement the ultraviolet light (UV) sensitivity of a E. coli recA deletion mutant. Thus, the thermophilic bacterium has a RecA protein whose function will be common to the E. coli RecA protein.View full abstractDownload PDF (382K) -
Kanoktip Packdibamrung, Haruo Misono, Makoto Harada, Shinji Nagata, Su ...1993 Volume 114 Issue 6 Pages 930-935
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn inducible NADP+-dependent D-phenylserine dehydrogenase [EC 1. 1. 1.-], which cata-lyzes the oxidation of the hydroxyl group of D-threo-β-phenylserine, was purified to homogeneity from a crude extract of Pseudomonas syringαe NK-15 isolated from soil. The enzyme consisted of two subunits identical in molecular weight (about 31, 000). In addition to D-threo-β-phenylserine, it utilized D-threo-β-thienylserine, D-threo-β-hydroxynor-valine, and D-threonine as substrates but was inert towards other isomers of β-phenyl-serine and threonine. It showed maximal activity at pH 10.4 for the oxidation of D-threo-β-phenylserine, and it required NADP+ as a natural coenzyme. NAD+ showed a slight coenzyme activity. The enzyme was inhibited by p-chloromercuribenzoate, HgCl2 and monoiodoacetate but not by the organic acids such as tartronate. The Michaelis constants for D-threo-β-phenylserine and NADP+ were 0.44mM and 29μM, respectively. The N-terminal 27 amino acids sequence was determined. It suggested that the NADP+-binding site was located in the N-terminal region of the enzyme.View full abstractDownload PDF (1378K) -
Yuji Mizuno, Mikiharu Yoshida, Hideko Yamamoto, Shunsaku Hirai, Eijiro ...1993 Volume 114 Issue 6 Pages 936-941
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the distributions of two known dystrophin isoforms derived from the 3' part of the dystrophin gene and of the dystrophin-associated proteins [50 DAG (A 2) and 43 DAG (A 3 a)] by immunoblot analysis, we examined various monkey tissues [skeletal (quad-riceps), cardiac (left ventricle), and smooth (aorta and uterus) muscles, lung, liver, central nervous system (cerebrum, cerebellum, and spinal cord), and peripheral nerve (sciatic nerve)]. With the antibody against the C-terminal domain of dystrophin, we found three bands other than the 400-kDa band corresponding to dystrophin or utrophin. These are a 130-kDa band detected in central nervous system, a 110-kDa band in peripheral nerve and spinal cord, and a 70-kDa band in all the tissues examined except skeletal muscle and peripheral nerve. The 130-kDa band may be a novel protein not yet reported, while the last two bands may correspond to Dp 116 and Dp 71, respectively [Lederfein et al. (1992) Proc. Natl. Acad. Sci. USA 89, 5346-5350 and Byers et al. (1993) Nature Genet. 4, 77-81]. Dystro-phin-associated protein, 43 DAG, was detected in all the tissues examined, but 50 DAG was detected only in skeletal and cardiac muscles. Furthermore, a unique 70-kDa band reacting with anti-50 DAG antiserum was found in all the tissues other than skeletal muscle.View full abstractDownload PDF (2155K)
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