All issues
Volume 99 (1986)
- Issue 6 Pages 1551-
- Issue 5 Pages 1289-
- Issue 4 Pages 993-
- Issue 3 Pages 607-
- Issue 2 Pages 315-
- Issue 1 Pages 1-
Volume 99, Issue 4
Displaying 1-32 of 32 articles from this issue
- |<
- <
- 1
- >
- >|
-
Matthias RÖNER, Peter GRÄBER1986 Volume 99 Issue 4 Pages 993-1003
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ATPase of the thermophilic bacterium PS3, TF0F1 and its subunits has been isolated and their absorption and fluorescence spectra have been measured. The following results were obtained:
(1) The tryptophan content of the subunits was determined spectroscopically.
(2) Although tryptophan (Trp) and tyrosine (Tyr) are found in TF1, the fluorescence spectrum of native TF1 and its subunits is dominated by Tyr fluorescence; this is in contrast to other proteins.
(3) Among (native) TF1 and its subunits only TF1 and the u-subunit show a weak fluorescence of Trp, which is blue-shifted, indicating a location in a strongly hydro-phobic environment.
(4) TF0 fluorescence is dominated by the strong Trp fluorescence.
(5) TF0F1 fluorescence is also dominated by the Trp residues. Additionally, its fluorescence is higher than the sum of the isolated TF0 and TF1, indicating marked changes in the microenvironment of the fluorescing aminoacids upon binding of TF1 to TF0.View full abstractDownload PDF (844K) -
Virinder K. MOUDGIL, Giuseppina LOMBARDO, Thomas EESSALU, Naomi ELIEZE ...1986 Volume 99 Issue 4 Pages 1005-1016
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA majority of the untransformed glucocorticoid-receptor complexes (GRc) from rat liver cytosol sedimented in the 9S region in 5-20% sucrose gradients containing 0.15M KCl and 20mM Na2MoO4. Incubation of the cytosol at 23°C, or at O°C with 10mM ATP or 0.3M KCl caused appearance of a slower migrating (4S) form which exhibited an increased affinity toward DNA-cellulose and ATP-Sepharose. Presence of 20mM Na2MoO4 blocked this 9S to 4S transformation of GRc. A complete conversion of the 9S to the 4S form occurred upon a 2h incubation of GRc with 10mM ATP at 0°C. Other nucleoside triphosphates (GTP, CTP, and UTP), ADP and PP1 (but not AMP or cAMP) were also effective in transforming the 9S form. The heat transformation occurred in a time-dependent manner and was complete within 1h at 23°C; presence of 10mM ATP during this 23°C incubation period allowed a complete 9S to 4S alteration in 10-20min. Addition of ATP also accelerated the rate of salt activation of the GRc; a 50% conversion to the 4S form occurred in 20min or 3min in the absence or the presence of 10 mM ATP during the 0°C incubation of GRc with 0.15 M KCl. An absolute requirement of the hormone for 9S to 4S transformation of glucocorticoid receptor (GR) was evident, as no conversion of the 9S form to the 4S form could be achieved with the ligand-free GR under any of the above conditions. Incubation of cytosol preparations at 23°C or at 0°C with KCl or ATP caused dissociation of the GRc and reduced the steroid binding capacity of GR. Although aurintricarboxylic acid, pyridoxal 5'-phosphate, Na2MoO4, Na2WO4, o-phenanthroline, Rifamycin AF/013 and heparin inhibited the ATP-Sepharose and DNA binding of the GRc, only Na2MoO4 and Na2WO4 selectively blocked the 9S to 4S conversion. We suggest that the 9S to 4S transformation in vitro of rat liver GRc represents an acquisition of DNA and ATP-Sepharose binding ability and may involve a separation of subunits from an oligomeric receptor structure.View full abstractDownload PDF (936K) -
Fujio NAGASHIMA, Sumio TANASE, Tsuyoshi HIGAKI, Yoshimasa MORINO, Sawa ...1986 Volume 99 Issue 4 Pages 1017-1025
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protease from Streptomyces violaceochromogenes (Murao, S., Nishino, Y., & Maeda, Y. (1984) Agric. Biol. Chem. 48, 2163-2166) is known to inactivate pig heart aspartate aminotransferase [EC 2.6.1.1]. Chemical analysis of the core proteins and peptide fragments produced upon proteolysis of the aminotransferase revealed that peptide bond cleavage occurred specifically at Leu 20 with concomitant inactivation. Neither inactivation nor peptide bond cleavage was observed with the mitochondrial isoenzyme. The proteolytically produced derivative 21-412 of the cytosolic isoenzyme retained approximately 0.1% enzymic activity for transami-nation with natural dicarboxylic substrates. The pyridoxal form of the derivative 21-412 was fully converted by cysteinesulfinate or alanine to the pyridoxamine form and conversely the pyridoxamine form of the derivative was also fully converted by 2-oxoglutarate or pyruvate into the pyridoxal form, indicating that the derivative was still catalytically competent. However, the rates of reaction with dicarboxylic substrates were much reduced whereas the rates with monocarboxylic substrates remained at an order of magnitude similar to that observed with the native enzyme. Thus the NH2-terminal segment appears to be an important structural component which determines the substrate specificity of aspartate aminotransferase for dicar-boxylic keto and amino acids. A substantial alteration in the molecular structure accompanying the loss of the NH2-terminal 20 residues was also reflected by the decrease in heat stability and in the lowering of the pKa value for His 68, which is involved in the intersubunit interaction of this dimeric enzyme.View full abstractDownload PDF (754K) -
Mikiharu YOSHIDA, Koichi YAGI1986 Volume 99 Issue 4 Pages 1027-1036
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA phosphatase that dephosphorylates myosin and the isolated light chain has been purified to near homogeneity from chicken gizzard smooth muscle. The molecular weight of the enzyme was estimated to be 100, 000 and 35, 000 under native and denatured conditions, respectively. It requires Mg2+ or Mn2+. The activity was measured quantitatively with a coupled enzyme system with the aid of myosin light chain kinase. The Vm and Km were determined to be 23.4μmol/mg/min and 4.2μm, respectively, with the isolated light chain as substrate under the optimal conditions (5mM Mg2+ at pH 8.45). The specific activity with myosin as substrate at a concentration of 0.9μm was found to be 1.25μmoiling/min, which was about one-fifth of the activity for the isolated light chain under the same conditions. The phosphatase seems to be specific to gizzard myosin. It may play an important role in the regulation of the myosin-actin interaction in smooth muscle.View full abstractDownload PDF (2181K) -
Maho KAWASHIMA, Shigekazu KITANI, Takeshi TANAKA, Takashi OBINATA1986 Volume 99 Issue 4 Pages 1037-1047
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monoclonal antibody (C-315) specific for cardiac-type C-protein was prepared and, in combination with other antibodies specific for fast and slow skeletal muscle C-proteins, it was used to investigate the expression of C-protein isoforms in developing striated muscle cells in vivo and in vitro. During embryonic development of skeletal muscles, a C-protein recognized by C-315 appeared first but only transiently, it being replaced subsequently by two other isoforms recognized by the antibodies to slow and fast skeletal muscle C-proteins in a fiber-type specific manner as previously demonstrated (Obinata et al. (1984) Develop. Biol. 101, 116-124). In contrast, only cardiac-type C-protein was detected in cardiac muscle throughout the develop-mental stages. When myogencsis in vitro was monitored using the same antibodies, C-315 binding appeared first in multinucleated myotubes as in vivo which was followed by the sequential expression of two other C-protein variants. The reactivity of C-315 as well as that of anti-slow and anti-fast skeletal C-protein antibodies persisted during muscle development in culture. Thus, this study demonstrates that the earliest form of C-protein expressed in striated muscles may either be a cardiac-type isoform or a unique embryonic protein containing an epitope in common with the adult cardiactype protein, and that transitions of C-protein isoform expression characteristic of each fiber-type occur during muscle development in vivo but not in vitro.View full abstractDownload PDF (4945K) -
Tomomitsu HATAKEYAMA, Nobuyuki YAMASAKI, Gunki FUNATSU1986 Volume 99 Issue 4 Pages 1049-1056
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nature of the saccharide-binding site of ricin D, which is a galactose- and N-acetylgalactosamine-specific lectin, was studied by chemical modification and spectroscopy. With excitation at 290nm, ricin D displayed a fluorescence spectrum with a maximum at 335nm. Upon binding of the specific saccharides, the spectrum shifted to shorter wavelength by 3nm. However, binding of galactosamine and N-acetylgalactosamine failed to induce such a change in the fluorescence spectrum. The interaction of ricin D with its specific saccharides was analyzed in terms of the variation of the intensity at 320nm as a function of saccharide concentration. The results indicate that the change in the fluorescence spectrum induced by sac-charide binding is attributable to the binding of saccharide to the low-affinity (LA-) binding site of ricin D.
The cytoagglutinating activity of ricin D decreased to 2% upon modification of two tryptophan residues/mol with N-bromosuccinimide at pH 4.0, but in the presence of galactose or lactose one tryptophan residue/mol remained unmodified, and a fairly high cytoagglutinating activity was retained. Galactosamine and N-acetylgalactosamine did not show such a protective effect. Spectroscopic analyses indicate that the decrease in the cytoagglutinating activity of ricin D upon tryptophan modification is principally due to the loss of the saccharide binding activity of the LA-binding site. The results suggest that one trypotphan residue is essential for saccharide binding at the LA-binding site, which can bind galactose and lactose but lacks the ability to bind N-acetylgalactosamine and galactosamine.View full abstractDownload PDF (651K) -
Nural ABSAR, Nobuyuki YAMASAKI, Gunki FUNATSU1986 Volume 99 Issue 4 Pages 1057-1062
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nature of the binding of specific saccharides to Abrus precatorius agglutinin (APA) was studied by ultraviolet difference spectroscopy. Upon binding of sac-charides, APA displayed difference spectra with maxima at 291-292 nm and 284-285 nm. Such spectra suggest that the state of the tryptophan residue closely associated with the saccharide-binding activity of APA is perturbed by the binding of a saccharide. The difference spectra value (Δε) increased with increasing saccharide concentration. From the increase in Δε at 291-292 nm, the association constant (Ka) was obtained for the binding of individual saccharides to APA. Lactose bound to APA with the highest affinity among the saccharides examined and its Ka value (8.3×103M-1 at pH 7.0 and 25°C) was approximately four times as large as that of galactose (2.2×103M-1). Raffinose and methyl β-galactopyranoside showed larger association constants than galactose. Galactosamine, N-acetyl-galactosamine and 2-deoxy galactose were found to bind with APA with fairly low affinity. The shape of the lactose-induced difference spectrum changed with pH and the spectrum in the acidic region showed characteristic broadening of the difference maximum peaks. The affinity of lactose to APA was nearly equal in the range of pH 6-8, but decreased outside this pH region and with increasing temperature.View full abstractDownload PDF (461K) -
Yuko ODA, Jun HIRABAYASHI, Ken-ichi KASAI1986 Volume 99 Issue 4 Pages 1063-1070
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMonoclonal antibodies against an endogenous β-galactoside-binding lectin (monomer molecular weight 14, 000, 14K lectin) of chick embryo were prepared and characterized. The inhibitory activities against hemagglutination, antigenic determinants and binding specificities were examined. Monoclonal antibody S1A4-5 strongly inhibited the hemagglutination activity of this lectin. This antibody did not bind to any cyanogen bromide (BrCN) fragment of the lectin. Another monoclonal antibody, S1A4-3, bound to one of the BrCN fragments (residues 34-66). How-ever, this antibody inhibited hemagglutination only weakly. The bindings to isolectins of β-galactoside-binding lectin, namely 14K lectin, 16K lectin (monomer molecular weight 16, 000) and a third species which is assumed to be a hybrid mol-ecule consisting of 14K and 16K lectin subunits, were examined. The antibody SIA4-5 bound to 14K lectin but not to 16K lectin. In the case of the third species, intermediate binding was observed.View full abstractDownload PDF (650K) -
Takuji MORIMOTO, Michiki KASAI1986 Volume 99 Issue 4 Pages 1071-1080
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSarcoplasmic reticulum (SR) vesicles were reconstituted by the salting out method in the presence of excess phospholipids: the lipid-to-protein ratio ranged from 10 to 100. It was found that the reconstituted vesicles could be separated by KCl density gradient centrifugation into four types: those having both cation and anion channels (CASR), those having only cation channels (CSR), those having only anion channels (ASR), and those having no ion channels (PSR). From the yield of these vesicles, it was estimated that one native SR vesicle contains 19 cation channels and 1.4 anion channels on average; the amount of cation channels is 14 times larger than that of anion channels. Although all vesicles thus prepared are considered to contain the Ca2+ -ATPase protein, the PSR vesicles alone did not take up Ca2+ but they did do so in the presence of valinomycin. This result indicates that the Ca2+ -ATPase takes up Ca2+ in an electrogenic manner. The electromotive force was estimated to be about 50mV.View full abstractDownload PDF (770K) -
Sei-ichi ISHIGURO, Hiroaki YAMAMOTO, Nobuaki YANAI, Takuji TAKEUCHI1986 Volume 99 Issue 4 Pages 1081-1085
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA sensitive, specific, competitive enzyme-linked immunosorbent assay (ELISA) was developed for quantitative analysis of tyrosinase. Binding sites of anti-tyrosinase antibodies were competed for by purified tyrosinase adsorbed onto microtiter plates and a known (standard) or unknown (sample) amount of tyrosinase in solution. Adsorbed antibodies were detected by goat anti-rabbit IgG F(ab')2 labeled with peroxidase. A sensitivity range of 2.1 to 14 ng (30-200 fmol)/well was obtained. SDS was found to be the most suitable detergent for solubilizing the enzyme. Tyrosinase was extracted from B16 mouse melanoma and assayed by the ELISA. The tyrosinase content per mg melanoma protein was 505±106 (S.D. ) ng. This assay is not only useful for measuring the content of normal tyrosinase in crude extracts but also is possibly applicable to detecting the unprocessed tyrosinases.View full abstractDownload PDF (353K) -
Kouji MATSUOKA, Kinuko KIMURA1986 Volume 99 Issue 4 Pages 1087-1100
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSReduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210, 000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160, 000 and 56, 000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210, 000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g=2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite-reduced states of the enzyme.
The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH-NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gin-dependent reactions coincided with the two-site ping-pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction de-viated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydro-genase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources.View full abstractDownload PDF (1891K) -
Yasuhiko ASADA, Yoshihiro SAWA, Katsuyuki TANIZAWA, Kenji SODA1986 Volume 99 Issue 4 Pages 1101-1110
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Kynurenine aminotransferase [L-kynurenine:2-oxoglutarate aminotransferase (cyclizing), EC 2.6.1.7] has been purified to homogeneity and crystallized from cell-free extracts of a yeast, Hansenula schneggii, grown in a medium containing L-tryptophan as an inducer. The enzyme has a molecular weight of about 100, 000 and consists of two subunits identical in molecular weight (52, 000). The enzyme exhibits absorption maxima at 280, 335, and 430 nm, and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. The enzyme-bound pyridoxal 5'-phosphate shows negative circular dichroic extrema, in contrast with other pyridoxal 5'-phosphate acting on L-amino acids. In addition to L-kynurenine and u-ketoglutarate, which are the most preferred substrates, a large number of L-amino acids and a-keto acids can serve as substrates; the extremely broad substrate specificity is the most char-acteristic feature of this yeast enzyme. The enzyme activity is significantly affected by both carbonyl and sulfhydryl reagents. Certain dicarboxylic acids such as adipate and pimelate act as competitive inhibitors. Addition of various substrate amino acids to the culture medium results in the inductive formation of aminotransferases which are immunochemically indistinguishable from L-kynurenine aminotransferase.View full abstractDownload PDF (1458K) -
Yoshifumi JIGAMI, Nobumasa TOSHIMITSU, Hirofumi FUJISAWA, Hiroshi UEMU ...1986 Volume 99 Issue 4 Pages 1111-1125
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSYeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of β-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENOI gene expression was found to be higher in stationary phase cells than in log phase cells.View full abstractDownload PDF (3235K) -
Tatzuo UEKI, Yoji INOKO, Mikio KATAOKA, Yoshiyuki AMEMIYA, Yuzuru HIRA ...1986 Volume 99 Issue 4 Pages 1127-1136
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSX-ray scattering profiles in moderate-angle regions were recorded from carbonmonoxy-, oxy-, and deoxyhemoglobin solutions, using synchrotron radiation. They all display four distinct scattering peaks at R=0.030, 0.055, 0.078, and 0.102Å-1 up to 2θ-10° in addition to the main scattering around R≈0. Contrast variation experiments, in which sucrose was used to change the electron density level of the solvent, revealed that the outer two scattering peaks are attributable to the variation of electron density within subunits in hemoglobin. The inner two were assigned as peaks due to the whole molecule and interpreted in terms of an interference function that is calculated from the inter-subunit distances in a molecule. This result is important in connection with evaluating the arrangement of constituent subunits in allosteric proteins and oligomeric proteins. The scattering profiles indicate that there is no difference in electron density variation within subunits between oxy- and deoxyhemoglobin. However, the arrangement of subunits is different between oxy-and deoxyhemoglobin molecules, as the scattering peaks at R=0.030 and 0.055Å-1 shift toward smaller angles for deoxyhemoglobin.View full abstractDownload PDF (756K) -
Shingo FUJISAKI, Tokuzo NISHINO, Hirohiko KATSUKI1986 Volume 99 Issue 4 Pages 1137-1146
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUpon rehydration of lyophilized Escherichia coli cells with phosphate buffer containing [14C]isopentenyl pyrophosphate (IPP), 14C was incorporated into the cells. Radioactivity was found in ubiquinone-8, an unidentified precursor of ubiquinone-8, demethylmenaquinone-8 and phosphate esters of all-trans-octaprenol and cis, trans-polyprenols. On rehydration of the cells with the buffer containing geranyl pyrophosphate or farnesyl pyrophosphate in combination with [14C]IPP, higher radio-activity was incorporated into the above products and some radioactivity was found in free prenols.
Fractionation of the 14C-labeled cells by sucrose-density gradient centrifugation before and after recultivation indicated that the size of 14C-Labeled cells had changed during the recultivation. This shows that radioactivity of [14C]IPP was incorporated into live cells but not into dead cells. The metabolism of the radioactive products in the recultivated cells was examined. It was found that the unidentified precursor was converted to ubiquinone-8, but demethylmenaquinone-8 was not converted to menaquinone-8. “Lipid intermediates” in peptidoglycan synthesis increased in the logarithmic growth phase and decreased in the stationary phase. In the stationary phase, however, an increase in cis, trans-polyprenyl monophosphates was observed. These observations suggest the operation of the lipid cycle of peptidoglycan synthesis.View full abstractDownload PDF (845K) -
Hiroshi SHIBATA, Saburo HARA, Tokuji IKENAKA, Jiro ABE1986 Volume 99 Issue 4 Pages 1147-1155
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20, 000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine α-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of α-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and α-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.View full abstractDownload PDF (1537K) -
Hiroshi SAKAI, Koichi SUZUKI, Kazutomo IMAHORI1986 Volume 99 Issue 4 Pages 1157-1167
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPyruvate kinase was purified to homogeneity from a moderate thermophile, Bacillus stearothermophilus. The molecular weight of the enzyme was found to be 250, 000 on gel filtration and 242, 000 on sedimentation analysis. The enzyme consisted of four identical subunits of a molecular weight of 62, 000-64, 000. There were no remarkable differences between the thermophilic enzyme and mesophilic enzymes in amino acid composition, secondary structure, mono- and di-valent cation require-ments for activity or specificity for nucleoside diphosphates. But the thermophilic enzyme was stable at high temperature and for a longer period of storage at lower temperature. Its specific activity was relatively high even at a low temperature (30°C).
The enzyme exhibited homotropic positive cooperativity for phosphoenol-pyruvate, but not for ADP. It was allosterically activated by AMP, ribose 5-phosphate and nucleoside monophosphates, but not by fructose 1, 6-bisphosphate. Activation by AMP and ribose 5-phosphate, and inhibition by inorganic phosphate were also observed even at the physiological temperature (60°C) for the thermophile.View full abstractDownload PDF (1454K) -
Yoshio INOKUCHI, Reiji TAKAHASHI, Tadaaki HIROSE, Seiichi INAYAMA, Ann ...1986 Volume 99 Issue 4 Pages 1169-1180
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase β-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and β-subunit proteins.
We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase β-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.View full abstractDownload PDF (934K) -
Takashi FURUSATO, Jun-ichi TAKANO, Yoshifumi JIGAMI, Hideaki TANAKA, K ...1986 Volume 99 Issue 4 Pages 1181-1190
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn 85 by DNA fragment, the nucleotide sequence of which had 84% homology with the sequence for the promoter, ribosome binding site and NH2-terminal five amino acids of the Bacillus amyloliquefaciens α-Amylase gene, was chemically synthesized. In order to analyze the promoter activity of a Bacillus subtilis α-Amylase secretion vector, the fragment was inserted between the promoter and signal peptidecoding region of Bacillus subtilis α-Amylase gene. Both promoters, tandemly repeated, functioned in transcribing the B. subtilis α-Amylase signal peptide-coding region followed by the Escherichia coli β-lactamase structural gene. The transcrip-tion initiation sites were determined by the primer extension method. The extracellular production of β-lactamase was stimulated by two promoters as compared with that by the plasmids containing either promoter region alone. The change of two amino acids in the NH2-terminal region of the B. subtilis α-Amylase signal peptide had no effect on the secretion of β-lactamase from B. subtilis cells.View full abstractDownload PDF (2055K) -
Masamichi IKEGUCHI, Kunihiro KUWAJIMA, Shintaro SUGAI1986 Volume 99 Issue 4 Pages 1191-1201
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComparative studies of the unfolding equilibria of two homologous proteins, bovine α-Lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of α-Lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein.
In previous studies, α-Lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of α-Lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, α-Lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the fea-tureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+ the unfolding transition of α-Lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native α-Lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.View full abstractDownload PDF (846K) -
Shiro KANEGASAKI, Ken-ichi TANAMOTO, Tatsuji YASUDA, J. Yuzuru HOMMA, ...1986 Volume 99 Issue 4 Pages 1203-1210
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the structure-activity relationships, various biological activities, including pyrogenicity, lethal toxicity, elicitation of Shwartzman reaction, mitogenicity and tumor necrosis factor (TNF)-inducing activity, were compared among natural and synthetic lipid A's differing in fatty acid composition. In all these tests, natural lipid A's from Escherichia coli and Salmonella minnesota and synthetic LA-l5-PP, which carries 3-hydroxy- and 3-acyloxy-tetradecanoyl groups at the 2, 3 and 2', 3' positions, respectively, showed the strongest activities among the tested lipid A's. In contrast, LA-16-PP, in which the amide-bound 3-hydroxytetradecanoic acid at position 2 of LA-15-PP is replaced by 3-hexadecanoyloxytetradecanoic acid, exhibited lower activity than LA-15-PP and natural lipid A's. Although LA-16-PP has been assumed to have a typical Salmonella lipid A structure (and, in fact, it has a structure corresponding to one of the components of Salmonella lipid A), the activity of this synthetic compound was not comparable to that of natural Salmonella lipid A. LA-17-PP, in which tetradecanoic acid is the sole fatty acid component, exhibited relatively strong mitogenicity and TNF-inducing activity, but very low pyrogenicity. The activities of LA-18-PP, which has ester-bound tetradecanoic acid and amide-bound 3-hydroxytetradecanoic acid, were lower than those of LA-17-PP. The results indicate that the differences in fatty acid composition of lipid A's have important influences on the biological activities studied.View full abstractDownload PDF (542K) -
Masaru TANOKURA, Harumi ARAMAKI, Kumi GOTO, Ukako HASHIMOTO, Yukiko TO ...1986 Volume 99 Issue 4 Pages 1211-1218
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo major isotypes of parvalbumins (PA1 and PA2) have been isolated from the skeletal muscle of bullfrog, Rana catesbeiana. The Mr values were estimated to be 10, 100 (PAl) and 11, 800 (PA2) by SDS/polyacrylamide gel electrophoresis, and the isoelectric points were determined to be 4.78 (PAl) and 4.97 (PA2) by polyacrylamide gel isoelectric focusing. The amino acid compositions and isoelectric points indicate that PA1 corresponds to Rana esculenta pI 4. 50 and Rana temporaria pI 4.75 parvalbumins and PA2 to Rana esculenta pI 4.88 and Rana temporaria pI 4.97 parvalbumins, showing that PA1 is genetically a β-parvalbumin and PA2 an α-parvalbumin. However, in terms of the amino acid compositions, PA1 and PA2 are distinctly different from the corresponding parvalbumins of Rana esculenta or Rana temporaria. The ultraviolet spectra of PAI and PA2 are consistent with their amino acid compositions. An ultraviolet difference spectrum of the Ca2+-loaded form vs. metal-free form indicates that a Tyr and some Phe residues in PAl are affected by a conformational change associated with the binding of Ca2+. On electrophoresis in polyacrylamide gel in 14mM Tris and 90mM glycine, the Ca2+-loaded form of PAl migrated twice as fast as the Mg2+-loaded form. Both PA1 and PA2 show increased mobility in the Ca2+-loaded forms, like troponin C but different from calmodulin.View full abstractDownload PDF (554K) -
Kumiko IKENO, Takeyuki IKENO, Hiroshi KUZUYA, Isao ISHIGURO1986 Volume 99 Issue 4 Pages 1219-1226
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA trypsin-like protease (named RSP-V) was purified to homogeneity from rat submandibular glands by isoelectric focusing and high-performance liquid chromatography. The purified enzyme had an isoelectric point of 5.3 and an apparent molecular weight of 25, 000, and consisted of two subunits with molecular weights of 19, 500 and 6, 000. RSP-V hydrolyzed BAEE, BAPA, and TAME, but not ATEE or BTPA. It had an optimum pH at around 10.0. RSP-V was strongly inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, but not by ovomucoid trypsin inhibitor, p-CMB, or iodoacetic acid. This enzyme partly resembled, but was not identical with, tonin. It was also different from kallikrein, salivain, and glandulain in rat submandibular gland. Although the physiological role of RSP-V has not yet been clarified, this enzyme inactivated dopa decarboxylase alone among catecholamine-synthesizing enzymes.View full abstractDownload PDF (532K) -
Kiyoshi KONISHI, Masato OUCHI, Kiyoshi KITA, Isamu HORIKOSHI1986 Volume 99 Issue 4 Pages 1227-1236
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cytochrome b560-d complex, a terminal oxidase in the respiratory chain of Photobacterium phosphoreum grown under aerobic conditions, was purified to near homo-geneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides with molecular weights of 41, 000 and 54, 000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 10.2 nmol of protoheme and 22.5 nmol of iron/mg of protein.
The enzyme is a “cytochrome bd-type oxidase, ” showing absorption peaks at 560 and 625nm in its reduced minus oxidized difference spectrum at 77K. This oxidase combined with CO, and its CO difference spectrum at room temperature in the Soret region showed a peak at 418nm and a trough at 434nm. In addition, a trough at 560nm (cytochrome b), and a trough at 620nm and a peak at 639nm (cytochrome d) were observed in the CO-binding spectrum.
This cytochrome b560-d complex catalyzed the oxidation of tibiquino1-1 and ascorbate in the presence of N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride or phenazine methosulfate. The oxidase activity required phospholipids and was inhibited by the respiratory inhibitors, KCN and NaN3, and the divalent cation, ZnSO4.
Formation of a membrane potential by the cytochrome b560-d complex reconstituted into liposomes was observed with the fluorescent dye, 3, 3'-dipropylthiodi-carbocyanine iodide, on the addition of ubiquinol-1, showing that the enzyme provided a coupling site for oxidative phosphorylation.View full abstractDownload PDF (732K) -
Noriaki KIKUCHI, Mitsuru SHIBATA, Mikiharu YOSHIDA, Koichi YAGI, Ichir ...1986 Volume 99 Issue 4 Pages 1237-1243
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMonoclonal antibodies against chicken breast myosin and its subfragment-1(S-1) were produced. One antibody, 2G41, reacted with S-1 containing a light chain 3 (LC3), but not with another S-1 containing a light chain 1 (LC1) or a mixture of the light chains. A structural difference can be assumed to exist between the head portions of the two myosin isozymes. Antigenicity of S-1 toward 2G41 could not be detected after tryptic digestion into three fragments of 50K, 27K, and 20K daltons.
Another monoclonal antibody, M68, was obtained from mice immunized with myosin. M68 preferably recognized the heavy chain from S-1 containing LC3 rather than that from that containing LC1 or S-1. M68 reacted with the 27K fragment among the three.View full abstractDownload PDF (532K) -
Kaoru OMICHI, Tokuji IKENAKA1986 Volume 99 Issue 4 Pages 1245-1252
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe modes of action of four α-amylase isozymes, which were purified from human saliva, on p-nitrophenyl α-maltopentaoside (G5P), maltohexaitol (G6R), and their 2-pyridylamino derivatives, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl- (1→4)-O-α-D-glucopyranosyl-(l→4)-O-α-D-glucopyranoside (FG5P) and O-6-deoxy-6-[(2-pyridy1)-amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyra-nosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucitol (FG6R) were examined at various pH values. No differences in their modes of action on the substrates was found. Irrespective of which enzyme was used, the molar ratio of the hydrolysis products of G5P or G6R was almost constant at any pH examined. On the other hand, those of FGSP and FG6R varied with pH such that predominantly O-6-deoxy-6[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucose (FG3) was formed at high pH ranges, while the formation of O-6-deoxy-6-[(2-pyridyl)amino]-α-D-gluco-pyranosyl(1→4)-O-α-D-gluco-pyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucose (FG4) increased at lower pH. The result indicates that the binding mode of FGSP or FG6R to the active sites of the enzymes changed with pH; namely, interactions between the 2-pyridyl-amino residue of the substrates and some amino acid residue(s) located in the active sites were influenced by pH. That the pH-dependence of action of the four iso-zymes was the same suggests that the parts of their active sites which contact the 2-pyridylamino residue are all composed of the same amino acid residues.View full abstractDownload PDF (480K) -
Yasunori KOZUTSUMI, Yoshihide NAKAO, Toshio TERAMURA, Toshisuke KAWASA ...1986 Volume 99 Issue 4 Pages 1253-1265
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLysosomal acid α-mannosidase from porcine kidney was found to contain mannose (4.8 %), galactose (0.9 %), fucose (0.5 %), N-acetylglucosamine (3.1 %), and mannose 6-phosphate (0.1 %). Approximately 50% of the total hexose of the oligosaccharide chains could be released by endo-β-N-acetylglucosaminidase-H (endo-H). They were predominantly neutral, oligomannoside-type oligosaccharides containing 5, 6, and 9 mannose residues, respectively, in the centesimal ratio of 36:25:34. 500-MHz 1H-NMR spectroscopy in conjunction with sequential exoglycosidase digestion of the reduced compounds revealed that each of the three fractions consisted of a single isomer only; the Man9 compound has the following structure:
D1 Manα(1-2) C Manα(1-2) 4 Manα(1-3)-
Manα(1-2) D2 Manα(1-3) A Manβ(1-4) 3 GlcNAcol 2
Manα(1-6) 4'
Manα(1-2) D3 Manα(1-6) B
The Man6-compound lacks Man residues D1, D2, and D3 while the Man5-compound lacks Man-C as well. In addition to the neutral ones, some (5%) phosphorylated oligomannoside-type oligosaccharides were obtained.
The endo-H resistant glycopeptides were subjected to hydrazinolysis. Approximately 60% of the oligosaccharides released by hydrazine were found to be of rather small size; their composition can be represented as Man2-3GIcNAc[Fuc]0-1GIcNAcol.View full abstractDownload PDF (883K) -
Yuki ITO, Yohsuke YAMASAKI, Nobuko SENO, Isamu MATSUMOTO1986 Volume 99 Issue 4 Pages 1267-1272
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel 63000 PW obtained by the same method with TSKge1 G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbony1-2-ethoxy-1, 2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin.View full abstractDownload PDF (445K) -
Kenji HANAOKA, Seiichi TAKASAKI, Akira KOBATA, Hideaki MIYAMOTO, Tetsu ...1986 Volume 99 Issue 4 Pages 1273-1276
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAsparagine-linked sugar chains were quantitatively released from hepatits B surface antigen on hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The released oligosaccharides were composed of two major sialylated components and a trace of a neutral component. From the results of the combination of sequential exoglycosidase digestion and methylation analysis, the structures of the acidic and the neutral components were deduced to be NeuAcα2→6Galβ1→4G1cNAcβ1→2Manα1→3(±NeuAca2 6Galβ1→4G1cNAcβ1→2Manαl 6)Manβ1→4G1cNActil→4GIcNAcOT and Galβ1→4G1cNAcβ→2Manα1→3(Galβ1→4G1cNAcβ1→2Manαl→6)Manβ1→4G1cNAcβ1→4G1cNAcOT, respectively.View full abstractDownload PDF (286K) -
Makoto KITO, Hiroshi NARITA, Hitoshi TAKAMURA, Hwa Jin PARK, Takao MAT ...1986 Volume 99 Issue 4 Pages 1277-1280
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe early breakdown of phosphatidylinositol 4, 5-bisphosphate in human platelets stimulated by a threshold concentration of either collagen or thrombin was inhibited by 5mM NaF through its inhibition of phospholipase C activity. However, 5mM NaF did not inhibit Ca2+ mobilization due to the stimuli from internal stores, but it did inhibit the influx of extracellular Ca2+ through its suppression of thromboxane A2 formation.View full abstractDownload PDF (341K) -
Shinobu IMAJOH, Hiroshi KAWASAKI, Koichi SUZUKI1986 Volume 99 Issue 4 Pages 1281-1284
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+-Activated neutral protease (CANP), that consists of 80K and 30K subunits, is converted to a low-Ca2+-requiring form by autolysis in the presence of Ca2+. Phosphatidylinositol greatly reduces the Ca2+-requirement for the autolysis of native CANP. However, this effect was not observed for CANP with a trimmed 30K subunit lacking the NH2-terminal hydrophobic and glycine-rich region. This suggests that the NH2-terminal hydrophobic region of the 30K subunit is important for the interaction of CANP with the cell membrane and that the calcium sensitivity of CANP is increased at the cell membrane through the effect of phosphatidylinositol.View full abstractDownload PDF (855K) -
Tohru NAKANO, Ayako TERAWAKI, Hitoshi ARITA1986 Volume 99 Issue 4 Pages 1285-1288
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing aequorin-loaded rat platelets stimulated with collagen, we found two phases of Ca2+ mobilization, one coinciding with a shape change and the other with aggregation, which have not yet been detected in quin2-loaded platelets. U46619, a stable analogue of prostaglandin H2, induced only a shape change and a concomitant rapid rise in the cytoplasmic ionized calcium concentration ([Ca12+]). However, upon addition of U46619 to platelets previously stimulated with collagen in the presence of indomethacin, a rapid increase in [Ca12+] and a shape change occurred, and, after about 1min, second increase in [Ca12+] and aggregation occurred. The actions of U46619 were inhibited by an antagonist for the thromboxane A2 (TXA2) receptor. These results suggest that the collagen-induced shape change is initiated by TXA2-induced Ca2+ mobilization, and aggregation is induced by the secondary Ca2+ mobi-lization induced by TXA2 and the occupation of the receptor by collagen.View full abstractDownload PDF (294K)
- |<
- <
- 1
- >
- >|