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Volume 103 (1988)
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Volume 103, Issue 6
Displaying 1-31 of 31 articles from this issue
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Tatsuya Moriyama, Hitoshi Takamura, Hiroshi Narita, Ken-ichi Tanaka, T ...1988 Volume 103 Issue 6 Pages 901-902
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe thromboxane A2 antagonist, ONO-3708, completely inhibited the increase in cytosolic free Ca2+ in human platelets during activation with collagen. Half-maximal Ca2+ release and influx required about 3 and 4 nM STA2, a stable thromboxane A2 mimetic, respectively. However, half maximal activation of phospholipase C required about 18 nM STA2. This suggests that thromboxane A2 directly causes Ca2+ mobilization without further activation of phospholipase C during activation of human platelets with collagen.View full abstractDownload PDF (233K) -
Matsuko Moriyasu, Masaaki Yamamoto, Naotaka Hamasaki1988 Volume 103 Issue 6 Pages 903-904
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe fructose 2, 6-bisphosphate concentrations in unwashed, washed, and leukocyte-free erythrocytes were compared. The concentration in washed red cells was 31±15 pmol per ml of cells (mean±S. D., n=6). The concentration in unwashed erythrocytes was at least twofold higher, but the value in washed red cells was not due to leukocyte contamination because it did not decrease further when washed cells were passed through an Imgard column, which would have removed any remaining leukocytes. No platelets were detected among the washed erythrocytes. Thus, the concentration in erythrocytes after washing was ascribed solely to these cells. The fructose 2, 6-bisphosphate concentration did not change when the glycolytic activity varied with pH, indicating that this compound is not involved in the regulation of carbohydrate metabolism in erythrocytes under these conditions.View full abstractDownload PDF (249K) -
Yoshiki Higuchi, Yukio Morimoto, Akio Horinaka, Noritake Yasuoka1988 Volume 103 Issue 6 Pages 905-906
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSingle crystals of human epidermal growth factor have been prepared from a polyethylene glycol solution and characterized by X-ray diffraction. The crystals grow in a space group of P21 with unit cell dimensions of a=32.7, b=32.5, c= 22.3 Å, and β=96.9°. They contain a single molecule per asymmetric unit and show better diffraction than 2.5 Å.View full abstractDownload PDF (1320K) -
Kazuto Kobayashi, Norio Kaneda, Hiroshi Ichinose, Fumio Kishi, Atsushi ...1988 Volume 103 Issue 6 Pages 907-912
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis. Recently, Grima et al. (Nature (1987) 326, 707-711) and we (Biochem. Biophys. Res. Commun. (1987) 146, 971-975; Nucleic Acids Res. (1987) 15, 6733) reported four similar but distinct mRNAs that encode human TH. These mRNAs are constant for the major part, but are distinguishable from one another as to the insertion/deletion of 12-bp and 81-bp sequences near the N-terminus. We isolated genomic clones encoding the human TH gene and determined the nucleotide sequence. The human TH gene is split into 14 exons. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Taking into consideration also the results of Southern blot analysis of human genomic DNA, we concluded that the four types of human TH mRNA are produced through alternative splicing from a single gene. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon, and the inclusion/exclusion of the second exon. We propose a possible secondary structure for the latter alternative splicing pathway.View full abstractDownload PDF (563K) -
Iwao Ohtsuki, Yoshimichi Onoyama, Fumie Shiraishi1988 Volume 103 Issue 6 Pages 913-919
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTroponin and its components or fragments were observed in an electron microscope by the use of the rotary shadowing technique. In freshly prepared troponin with low viscosity, globular particles were mainly observed. The size of the long axis of the particles was 13.2±1.3 nm and the size perpendicular to the long axis was 9.5±1.2 nm. The mean axial ratio was 1.4±0.3. Most of the particles observed in a stored troponin preparation, having a higher viscosity than that of fresh troponin, had a globular head with a thin tail, with the total length of 25.4±1.4 nm (head-tail type particles). The axial size of the globular portion was 8.3±1.2 nm and the tail length was 17.1±1.6 nm. Observation of various particles during the transitional stages indicated that, in the globular particles, the tail region of head-tail type particle was associated along the globular head region. Troponin T was a filamentous particle with 16.9±1.5 nm length. The 26 K fragment of troponin T, which was devoid of the N-terminal 45 residues from troponin T, was a filamentous particle with the length of 14.4±1.3 nm. Troponin T1, one of two chymotryptic subfragments of troponin T, was a filamentous particle of 11.6±1.4 nm length. Troponin C•T in the presence of Ca2+ was a particle with a globular head (7 nm in size) and a tail of about 17 nm length. The Fab fragment of anti-troponin T1 formed regular transverse striations along the thin filament of rabbit skeletal muscle with a 38 nm period. The distance between the first striation and the top of the filament was less than one period length.View full abstractDownload PDF (4688K) -
Takayuki Nemoto, Yuko Ohara-Nemoto, Minoru Ota1988 Volume 103 Issue 6 Pages 920-927
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe [3H] triamcinolone acetonide ([3H] TA)-binding ability of the rat liver glucocorticoid receptor (GR) was investigated under acidic conditions, ranging from pH 2 to 7.3. Both in the presence and absence of 10mM molybdate, the [3H] TA-binding ability decreased below pH 6.5 and was almost completely lost below pH 5, pH 5.9±0.1 giving 50% [3H] TA-binding. The binding ability was recovered when the pH of the cytosol was reversed to 7.3 or the precipitate obtained on acidification was dissolved in a buffer of pH 7.3. Moreover, in the absence of molybdate, the [3H]TA-GR complexes formed at pH 7.3 remained unchanged until pH 5. Then they decreased, pH 3.9±0.1 giving 50% binding, and completely disappeared at pH 3. [3H] TA-binding activity recovered from the precipitate also decreased in a similar pH region (a 50% decrease in binding being observed at pH 4.2±0.04). These results suggest that rat liver GR is rather resistant under acidic conditions and that it exists in a peculiar state below pH 5.9 to _??_4 as to its ligand binding property: unoccupied GR has no [3H] TA-binding ability but [3H] -GR complexes once formed at neutral pH do not dissociate. [3H] TA-GR complexes recovered from the precipitate at pH 5 had a Stokes radius of 7.5 nm, little DNA-cellulose-binding ability and sedimented at 8.6 S on glycerol gradient centrifugation, indicating that the receptor existed in a nontransformed state. In addition, both occupied and unoccupied GR were transformed at about pH 4, their being 50% transformation. This transformation was accompanied by irreversible denaturation of the receptor. Molybdate prevented the acid-induced transformation and denaturation of both occupied and unoccupied GR. These results suggest that two kinds of dissociable groups, with pK= 5.9 and _??_4, respectively, are involved in the conversion between GR states. The former is related to the binding of [3H] TA, and the latter to the transformation and denaturation of GR. The most probable candidate for the dissociable group with pK= 5.9 is a histidyl residue at or near the steroid binding site, and for those with pK=_??_4 are acidic amino acid residues on a nonhormone-binding component of the nontransformed GR, i.e., heat shock protein 90.View full abstractDownload PDF (974K) -
Tsuneaki Koike, Kenzo Ohtsuki1988 Volume 103 Issue 6 Pages 928-937
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA nonhistone chromatin protein (NHCP) has been purified to homogeneity from a 0.5M NaCl extract of Ehrlich ascites tumor cell (EAT cell) nuclei as a phosphate acceptor for casein kinase II using ion-exchange column chromatographies and Sephacryl S 300 gel filtration. The purified NHCP (approximate Mr= 400, 000) was found to be a tetramer of an Mr= 98, 000 polypeptide (pI= 6.9) and to have high contents of glycine (15%) and serine (11.6%). This protein (designated as 400-kDa NHCP) was highly phosphorylated by casein kinase II (Mr= 130, 000), but not by histone kinase. Casein kinase II phosphorylated only seryl residues of the purified 400-kDa NHCP. The NHCP bound with DNA, but not with RNAs, and the DNA binding ability of the protein was reduced when it was phosphorylated by casein kinase II. Moreover, we found that (a) the 400-kDa NHCP is present in large quantities in malignant mouse cells, such as EAT, EL-4, and Meth-A cells, but only slightly in normal tissues and cells; (b) the protein level is rapidly increased when mouse lymphocytes are treated with recombinant interleukin 2 (T cell growth factor) or concanavalin A; and (c) the kinase responsible for the 400-kDa NHCP phosphorylation in the chromatin of various mouse cells is a casein kinase II. These experimental results suggest that the 400-kDa NHCP acts as an effective phosphate acceptor for casein kinase II at the chromatin level and that an increased phosphorylation of the protein by the kinase may be implicated in the progress of cell differentiation and proliferation.View full abstractDownload PDF (8983K) -
Masayoshi Takeuchi, Naoki Asano, Yukihiko Kameda, Katsuhiko Matsui1988 Volume 103 Issue 6 Pages 938-943
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSFlavobacterium saccharophilum cytoplasmic membranes contain several cytochromes linked to the respiratory chain. The presence of c-type cytochrome, cytochrome o, and a small amount of α-type cytochrome was proved. Cytochrome c551 was purified to electro-phoretic homogeneity by ion-exchange chromatography and gel filtration from a membrane fraction of F. saccharophilum and its properties determined. Cytochrome c551 possessed absorption peaks at 407 nm in the oxidized form, and at 415, 521, 551 nm in the reduced form. The cytochrome c551, had a molecular weight of 15, 500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Glucoside 3-dehydrogenase of F. saccharophilum reduced the cytochrome c551 with methyl-α-D-glucoside, D-glucose, sucrose, or validoxylamine A. When the purified glucoside 3-dehydrogenase was incubated with methyl-α-D-glucoside and purified ferricytochrome c551, methyl-α-D-3-ketoglucoside was formed as indicated by GC-MS analysis. The addition of a substrate to the membrane fraction caused an increase in the rate of oxygen uptake and an abrupt reduction in cytochrome c551. The electron transfer in the 3-keto sugar forming system may be as follows: sugars→glucoside 3-dehydrogenase→cytochrome c551, _??_cytochrome oxidase→O2. Thus, the electron acceptor of glucoside 3-dehydrogenase is possibly connected to the membrane-bound cytochrome system.View full abstractDownload PDF (1874K) -
Yoshinobu Kimura, Sumihiro Hase, Yuji Kobayashi, Yoshimasa Kyogoku, To ...1988 Volume 103 Issue 6 Pages 944-949
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe toxic lectin, ricin D, contains mannose, fucose, xylose, and N-acetylglucosamine as sugar components. Sugar chains are linked to Asn-10 of the A-chain, and to Asn-95 and Asn-135 of the B-chain (Funatsu, G. et al. (1978) Agric. Biol. Chem. 42, 501-503; Araki, T. & Funatsu, G. (1985) FEBS Lett. 191, 121-124). Asparagine-linked sugar chains of each glycopeptide from ricin D were liberated by hydrazinolysis followed by N-acetylation. The reducing end residues of the sugar chains were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives obtained were purified by gel-filtration and reversed-phase HPLC. Eight main PA-sugar chains were obtained from three glycopeptides and the structures of these sugar chains were determined by component analysis, stepwise exoglycosidase digestions, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. The results show that oligomannose type sugar chains (Man6-7, GlcNAc2) are linked to Asn-95; GlcNAc2 and M4X (structure, see below) to Asn-135 of the B-chain, and M3FX and M3X to Asn-10 of the A-chain.View full abstractDownload PDF (597K) -
Tomiko Asano, Nobuko Kamiya, Rika Morishita, Kanefusa Kato1988 Volume 103 Issue 6 Pages 950-953
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAntibodies were raised in rabbits against the βγ subunits of bovine brain GTP-binding proteins, and were purified with a βγ-coupled Sepharose column. Purified antibodies reacted strongly with 36, 000-dalton β subunit and slightly with 35, 000-dalton β and γ subunits, but not with other proteins in an immunoblot assay. Using these purified antibodies, a sensitive enzyme immunoassay method for the quantification of brain βγ was developed. The assay system consisted of polystyrene balls with immobilized antibody F (ab')2 fragments and the same antibody Fab' fragments labeled with β-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 3 fmol, or 130 pg. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1M NaCl, and the concentrations of βγ were determined. The βγ were detected in all the regions, and the highest concentrations were observed in the cerebral cortex and nucleus caudatus. The concentrations of βγ were higher than those of α subunit of GTP-binding protein, Go, in all the regions.View full abstractDownload PDF (2946K) -
Yoshikazu Tanaka, Toshihiko Ashikari, Yuji Shibano, Teruo Amachi, Haji ...1988 Volume 103 Issue 6 Pages 954-961
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.View full abstractDownload PDF (881K) -
Hirozo Oh-oka, Yasuhiro Takahashi, Kazumi Kuriyama, Kazuhiko Saeki, Hi ...1988 Volume 103 Issue 6 Pages 962-968
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe 9 kDa polypeptide from spinach photosystem I (PS I) complex was isolated with iron-sulfur cluster (s) by an n-butanol extraction procedure under anaerobic conditions. The polypeptide was soluble in a saline solution and contained non-heme irons and inorganic sulfides. The absorption spectrum of this iron-sulfur protein was very similar to those of bacterial-type ferredoxins. The amino acid sequence of the polypeptide was determined by using a combination of gas-phase sequencer and conventional procedures. It was composed of 80 amino acid residues giving a molecular weight of 8, 894, excluding iron and sulfur atoms. The sequence showed the typical distribution of cysteine residues found in bacterial-type ferredoxins and was highly homologous (91% homology) to that deduced from the chloroplast gene, frxA, of liverwort, Marchantia polymorpha. The 9 kDa polypeptide is considered to be the iron-sulfur protein responsible for the electron transfer reaction in PS I from center X to [2 Fe-2 S] ferredoxin, namely a polypeptide with center (s) A and/or B in PS I complex. It is noteworthy that the 9 kDa polypeptide was rather hydrophilic and a little basic in terms of the primary structure. A three-dimensional structure was simulated on the basis of the tertiary structure of Peptococcus aerogenes [8 Fe-8 S] ferredoxin, and the portions in the molecule probably involved in contacting membranes or other polypeptides were indicated. The phylogenetic implications of the structure of the present polypeptide as compared with those of several bacterial-type ferredoxins are discussed.View full abstractDownload PDF (2121K) -
Taichi Usui, Takeomi Murata1988 Volume 103 Issue 6 Pages 969-972
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTransglycosylation from maltopentaose to the 4-position of p-nitrophenyl α-glucoside was efficiently induced through the use of maltotetraose-forming amylase from Pseudomonas stutzeri in an aqueous solution containing methanol at a high concentration. The enzyme specifically formed p-nitrophenyl α-maltopentaoside (12% of the enzyme-catalyzed net decrease of maltopentaose) from maltopentaose as a donor and p-nitrophenyl α-glucoside as an acceptor. The rate of the transglycosylation depended on the concentration of methanol solvent, the pH and the temperature. Use of the aqueous methanol system in this reaction not only ensured a sufficient solubility of p-nitrophenyl α-glucoside but also resulted in a remarkable increase in the formation of p-nitrophenyl α-maltopentaoside, which is a useful substrate for assay of human amylase in serum and urine.View full abstractDownload PDF (419K) -
Yuji Naitoh, Shigeru Taketani, Rikio Tokunaga, Yoshiko Sameshima1988 Volume 103 Issue 6 Pages 973-978
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTo clarify the mechanisms involved in the specific uptake of hematoporphyrin by cancer cells, we investigated the interaction of the heme- and/or hematoporphyrin-hemopexin complexes with rat hepatoma dRLh-84 cells. Hemopexin bound to the cells in a saturable, time- and temperature-dependent manner. The cells exhibited 0.55 nmol of binding sites/mg of protein for the heme-hemopexin complex and 0.38 nmol for the hematoporphyrin-hemopexin complex. The dissociation constants (Kd) for the heme-hemopexin and hematoporphyrin-hemopexin complexes were 0.57 and 0.54 μM, respectively. Specific binding of the labeled hemopexin was inhibited by the unlabeled heme- and hematopor-phyrin-hemopexin complexes but was unaffected by albumin or neoglycoprotein. Hemato-porphyrin bound to hemopexin was incorporated into the cells at 37°C, but not at 4°C. These results indicate that hematoporphyrin bound hemopexin was taken up by dRLh-84 cells, via the hemopexin receptors. When the hematoporphyrin-albumin complex was incubated with the cells, the hematoporphyrin-[125I] albumin complex bound to the cells in a time and temperature-dependent manner. Here the binding was not saturated up to 100 μg/ml of albumin. The binding of hematoporphyrin-[125I] albumin was partially inhibited by unlabeled albumin and hemopexin. Hematoporphyrin bound to albumin was taken up by the cells at 37°C. Thus, the albumin-dependent uptake of hematoporphyrin by rat hepatoma dRL-84 cells could be differentiated from the hemopexin-mediated uptake of hematopor-phyrin.View full abstractDownload PDF (723K) -
Kiyoshi Uchida, Yuko Susai, Eri Hirotani, Takuma Kimura, Takaaki Yoney ...1988 Volume 103 Issue 6 Pages 979-985
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSResonance Raman spectra of myoglobin (Mb) reconstituted with 4-vinyl and 2, 4-divinyl deuterated protoheme IX were studied in several oxidation, spin, and ligation states (deoxyMb, MbO2, MbCO, metMbH2O, and metMbCN-) with special attention to the low frequency vibrations. Frequency shifts observed on deuteration enabled us to assign some Raman bands to vibrations specifically involving the 2- or 4-vinyl group. The most significant deuteration effect was found for a band around 410cm-1 in the ferrous state, which loses intensity on 4-vinyl deuteration and is ascribed to a porphyrin in-plane vibration strongly coupled with the skeletal bend of the vinyl group at position 4. Such strong coupling implies that the vinyl group lies in the same plane as the pyrrole II ring, as in the crystalline state. Thus, frequency shifts on vinyl deuteration may be useful as a probe of the orientation of the vinyl group. Resonance Raman spectra of Mb coordinated with various sizes of isocyanides are interpreted in terms of vinyl orientational changes induced by ligation.View full abstractDownload PDF (855K) -
Shunji Natsuka, Masaru Himeno, Sumihiro Hase, Harunobu Ito, Tadashi Ue ...1988 Volume 103 Issue 6 Pages 986-991
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe structures of sugar chains of a p-nitrophenyl acetate-hydrolyzing esterase from the microsomes of rat liver were established. The enzyme contained mannose and glucosamine as sugar components. Asparagine-linked sugar chains of the esterase were liberated by hydrazinolysis. After N-acetylation of the hydrazinolysate, the reducing ends of the sugar chains were coupled with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Eleven PA-sugar chains were obtained. The structures of the PA-sugar chains were first identified by HPLC using two series of separation systems by which 11 PA-oligomannosetype sugar chains with known structures could be separated. Further elucidation of the structures of each PA-sugar chain was performed by exoglycosidase digestions and partial acetolysis. The structures of two of the PA-sugar chains were further confirmed by 500 MHz 1H-NMR spectroscopy.View full abstractDownload PDF (1537K) -
Masayuki Nashimoto, Kikuo Ogata, Yukio Mishima1988 Volume 103 Issue 6 Pages 992-997
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWhen we compared the sequences near four terminal sites of transcripts including two endoribonucleolytic cleavage sites in external transcribed spacer 1 upstream of the 5' end of the mouse 18 S rRNA (Mishima, Y., Mitsuma, T., & Ogata, K. (1985) EMBO J. 4, 3879-3886), a seven-nucleotide consensus sequence, GGPyUUGPy (Py is C or U), was obtained. The results of both in vitro pulse-chase experiments and S 1 nuclease mappings using the mouse rDNA fragment of the transcription initiation region indicated that ribonucleolytic cleavages take place in the sequence matching the consensus sequence at more than five nucleotides out of the seven positions. Furthermore, effective ribonucleolytic cleavages in vitro were observed in a sequence, GGCUUGU, in the internal transcribed spacer 2 located between 5.8 S and 28 S rRNA. These results demonstrate that the ribonucleolytic cleavages occur preferentially in the sequence of GGPyUUGPy in the transcribed spacer regions of the mouse pre-rRNA. From this information, we infer the existence of processing steps for the pre-rRNA maturation involving the sequence-specific ribonucleolytic cleavages.View full abstractDownload PDF (3164K) -
Takao Taki, Miki Takamatsu, Akio Myoga, Koichi Tanaka, Susumu Ando, Ma ...1988 Volume 103 Issue 6 Pages 998-1003
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSGlycolipids of metastatic tissue in liver from colon cancer were separated by HPLC and their structures were analyzed. Neutral glycolipid was composed of five major components. Four of them showed similar mobilities to GlcCer, LacCer, Gb3Cer, and nLc4Cer on TLC. The structure of the fifth major component was analyzed by exo-glycosidase treatments, 400 MHz proton NMR spectrometry, gas chromatography-mass spectrometry of the methylated sugars, and immunostaining on a TLC-plate, and concluded to be
_??_ Ganglioside was composed of two major components. One of them was identical with GM3. The structure of the second ganglioside was analyzed by exo-glycosidase treatment, immunostaining on a TLC-plate, and gas chromatography-mass spectrometry of methylated sugars, and concluded to be
_??_ These LeX and sialyl-LeX structure-containing lipids accounted for about 12% of the neutral glycolipid fraction and 42% of the ganglioside fraction, respectively. From these results, it appears that the biosynthetic pathway for LeX structure-containing lipids is activated in colon cancer.View full abstractDownload PDF (4058K) -
Yoshiyasu Yabusaki, Hiroko Murakami, Hideo Ohkawa1988 Volume 103 Issue 6 Pages 1004-1010
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P 450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage λ gt 11. The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76, 737 daltons. The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase. In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase. The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases. In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.View full abstractDownload PDF (2200K) -
Hiroyuki Arata, Yasuharu Serikawa, Ken-ichiro Takamiya1988 Volume 103 Issue 6 Pages 1011-1015
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSErythrobacter sp. OCh 114, an aerobic photosynthetic bacterium, had trimethylamine N-oxide (TMAO) reductase activity, which increased when the culture medium contained TMAO. The reductase was located in the periplasm. The bacteria grew anaerobically in the presence of TMAO. These results suggested that Erythrobacter OCh 114 has the ability to reduce TMAO through the respiratory chain. The TMAO respiration system of this organism was different from those of facultative purple photosynthetic bacteria in two respects: (a) TMAO reductase did not have activity to reduce dimethyl sulfoxide and (b) soluble c-type cytochrome, cytochrome c551, and cytochrome b-c1 complex appeared to be involved. The photochemical activity, which is usually inoperative in the anaerobic cell suspension, was restored by TMAO, suggesting that the photosynthesis and the TMAO respiration share a common electron transfer chain.View full abstractDownload PDF (612K) -
Tetsuhiko Yoshimura, Hidekazu Iwasaki, Sohsuke Shidara, Shinnichiro Su ...1988 Volume 103 Issue 6 Pages 1016-1019
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSElectron paramagnetic resonance (EPR) spectra at 77 K have been measured for the dried cells of three groups of chemoheterotrophic bacteria cultured in the presence of nitrate: I, denitrifying bacteria containing cytochrome c' (Alcaligenes sp. NCIB 11015, Achromobacter xylosoxidans GIFU 543); II, denitrifying bacteria in which cytochrome c' has not been found (Alcaligenes faecalis IAM 1015, Bacillus firmus NIAS 237, Pseudomonas stutzeri ATCC 17641); III, non-denitrifying bacterium containing cytochrome c' (A. xylosoxidans GIFU 1055). A characteristic three-line EPR signal was detected only in the dried cells of group I, and was assigned to NO-cytochrome c' complex. The probable physiological role of the cytochrome c' in denitrifying bacteria is discussed.View full abstractDownload PDF (393K) -
Keisuke Mashima, Takashi Kimura, Weida Huang, Kazuo Yano, Yoshiyuki As ...1988 Volume 103 Issue 6 Pages 1020-1026
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIt was found that a non-tumorigenic epithelial cell line from the liver of a Buffalo-strain rat (BRL) secreted into the culture medium various inhibitors of the growth of BRL and RSV-BRL (tumorigenic BRL transformed by infection of Rous sarcoma virus). The secreted inhibitors were classified into two types: one inhibited the growth of BRL to a greater extent than that of RSV-BRL (non-tumorigenic BRL growth inhibitor, NGI), and the other, vice versa (tumorigenic BRL growth inhibitor, TGI). Two NGI (NGI-I and NGI-II) and two TGI (TGI-I and TGI-II) were highly purified from the serum-free conditioned medium. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis without 2-mercaptoethanol, NGI-I and II gave protein bands with molecular weights (Mr) of 56, 000 and 21, 000, respectively. TGI-I and II gave a band that migrated faster than bromophenol blue marker dye, but they did not pass through an ultrafiltration membrane with an Mr cutoff of 5, 000. In the presence of a reducing reagent, only NGI-II showed a decrease of Mr, from 21, 000 to 11, 000. NGI and TGI showed 50% growth inhibition with BRL and RSV-BRL, respectively, at 5-15 ng/ml in the medium containing 10% fetal calf serum. NGI and TGI all were stable to 1M acetic acid (pH 2.3) and 6M urea, but labile to 5mM dithiothreitol or trypsin. Of the eight cell lines tested, NGI-I was most effective on BRL, NGI-II on BRL and HSC-3 (human tongue squamous carcinoma), and both TGI-I and II on RSV-BRL.View full abstractDownload PDF (2913K) -
Akira Hara, Yoshio Inoue, Makoto Nakagawa, Fumiharu Naganeo, Hideo Saw ...1988 Volume 103 Issue 6 Pages 1027-1034
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA monomeric 3α-hydroxysteroid dehydrogenase with a molecular weight of 34, 000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3α-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and Δ4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP (H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A, were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme•NADPH complex and to the enzyme NADP+ complex.View full abstractDownload PDF (1996K) -
Tamao Endo, Ryuichiro Nishimura, Matsuto Mochizuki, Naohisa Kochibe, A ...1988 Volume 103 Issue 6 Pages 1035-1038
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe two subunits of human chorionic gonadotropin (hCG) purified from the urine of a patient with choriocarcinoma were successfully separated by SDS-polyacrylamide gel electrophoresis. A comparative study of the oligosaccharides released from the two subunits by hydrazinolysis revealed that altered glycosylation occurs in both subunits and possibly at all four asparagine sites of the choriocarcinoma hCG moleculeView full abstractDownload PDF (1478K) -
Purification and Characterization of 17β-Hydroxysteroid Dehydrogenase from Cylindrocarpon radicicolaEiji Itagaki, Teturou Iwaya1988 Volume 103 Issue 6 Pages 1039-1044
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAn NAD+-linked 17β-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58, 600 by gel filtration and polyacrylamide gel electropho-resis. SDS-gel electrophoresis gave Mr=26, 000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17β-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax=85 μmol/min/mg; Km for the steroid =9.5 μM; Km for NAD+=198 μM at pH 10.0) and for the reduction of androstenedione (Vmax=1.8 μmol/min/mg; Km for the steroid=24 μM; Km for NADH=6.8 μM at pH 7.0). In the purified enzyme preparation, no activity of 3α-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, Δ5-3-ketosteroid-4, 5-isomerase, or steroid ring A-Δ-dehydrogenase was detected. Among several steroids tested, only 17β-hydroxysteroids such as testosterone, estradiol-17β, and 11β-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17α-3H] testosterone.View full abstractDownload PDF (1897K) -
Hae-ik Rhee, Kousaku Murata, Akira Kimura1988 Volume 103 Issue 6 Pages 1045-1049
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSL-Alanine:4, 5-dioxovalerate aminotransferase, which catalyzes transamination between L-alanine and 4, 5-dioxovalerate to yield δ-aminolevulinate and pyruvate, has been purified from Pseudomonas riboflavina IFO 3140. The enzyme had a molecular weight of 190, 000 and consisted of four identical subunits. It was crystallized as pale yellow needles. The enzyme used L-alanine (relative activity 100), β-alanine (39), and L-ornithine (14) as amino donors. γ-Aminobutyrate (55) and ε-aminocaproate (34) were also effective as amino donors. The reaction proceeded according to a ping-pong mechanism and the Km values for L-alanine and 4, 5-dioxovalerate were 1.7 and 0.75mM, respectively. The activity of the enzyme is strongly inhibited by pyruvate, hemin, and methylglyoxal. Methylglyoxal interacted with the enzyme and brought about a complete inactivation.View full abstractDownload PDF (1677K) -
Susumu Ohara, Kazuhiko Ishihara, Kyoko Hotta1988 Volume 103 Issue 6 Pages 1050-1053
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSGastric mucus glycoproteins were extracted with 2% Triton X-100 from rat gastric corpus and antrum and purified by CsCl equilibrium centrifugation. Corpus mucus glycoproteins were degraded into what appeared to be two “subunits” (Mw 4.4×105 and 6×106) by the reduction of disulfide bonds. Papain digestion of the latter produced glycopeptides with a molecular weight of approximately 4.4×105. This type of subunit had carbohydrate chains with about 9 sugars attached to every 2 amino acid residues. Papain digestion of the former type of subunit revealed no change in the elution profile on Bio-Gel A-15m. This type of subunit had carbohydrate chains with 17-19 sugars attached to every 3 amino acid residues. The subunit of antral mucus glycoproteins was essentially the same as the former type of corpus subunits in molecular weight (Mw 4.4×105) and average oligosaccharide chain length. These results suggest that there are two distinct types of mucus glycoprotein subunits in rat stomach.View full abstractDownload PDF (370K) -
Etsuo Okuno, Robert J. White, Robert Schwarcz1988 Volume 103 Issue 6 Pages 1054-1059
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSQuinolinic acid phosphoribosyltransferase (QPRT) [EC 2. 4. 2. 19] from human liver and brain was purified to homogeneity. Identity of the pure enzymes isolated from the two organs was proven by biochemical, physicochemical and, following the production and partial purification of anti-liver QPRT antibodies, immunological techniques. Human QPRT has a molecular weight of 170, 000 and consists of five identical subunits. Kinetic analyses revealed a Km of 5.6 μM for the substrate (quinolinic acid) and 23 μM for the co-substrate (phosphoribosylpyrophosphate). Enzyme activity was dependent on Mg2+ (optimal concentration: 1mM) and was inhibited by the enzymatic by-product, inorganic pyrophosphate. Pure QPRT and its antibodies will constitute useful tools in the examination of the possible role of quinolinic acid in the pathogenesis of human neurodegenerative disorders.View full abstractDownload PDF (2544K) -
Tadashi Yoshimoto, Hiroshi Oyama, Takashi Honda, Hiroshi Tone, Tomoaki ...1988 Volume 103 Issue 6 Pages 1060-1065
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1, 426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-celluloseView full abstractDownload PDF (3118K) -
Hideo Oe, Etsushiro Doi, Masaaki Hirose1988 Volume 103 Issue 6 Pages 1066-1072
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe amino- (N-) and carboxyl- (C-)terminal half-molecules of ovotransferrin were pre-pared by a novel procedure. The trypsin-nicked ovotransferrin (Ikeda et al. (1985) FEBS Lett. 182, 305-309), in which the two half-molecules interact non-covalently forming a stable dimer, was purified by gel filtration and anion-exchange column chromatography. By subsequent cation-exchange chromatography, the nicked form was distinctly separated into an equivalent amount of the N-terminal and C-terminal half-molecules. Analyses of the N-terminal and C-terminal sequences indicated that the N-terminal and C-terminal half-molecules comprised the alignments of residues 1-332 and 342-686 of ovotransferrin, respectively. Anion-exchange chromatography, gel filtration chromatography, and nondenaturing polyacrylamide gel electrophoresis revealed that the isolated half-molecules had the ability to re-associate in solution. The contents of α-helix and β-sheet of the two half-molecules, as determined by circular dichroism (CD) spectra, were very similar to those of intact ovotransferrin. No prominent alteration in the secondary structure of the two half-molecules was induced by the re-association.View full abstractDownload PDF (3070K) -
Michiaki Morohashi, Katsutoshi Konishi, Masaru Kawamura1988 Volume 103 Issue 6 Pages 1073-1077
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe membrane bound (Na, K)-ATPase prepared from Artemia salina nauplii was solubilized with a zwitterionic detergent, 3[3(cholamidopropyl)dimethylammonio]-1-pro-panesulfonate (CHAPS), and then purified on a Bio-Gel A-1.5 m column in the presence of the detergent. 1) Upon solubilization, both NaCI and KCl protected the enzyme against loss of activity, KCl being more effective than NaCl. 2) Gel filtration of the solubilized enzyme on a Bio-Gel A-1.5m column in the presence of 5mM CHAPS resulted in loss of the enzyme activity even when one of the cations was added. Most of the phospholipids in the solubilized enzyme preparation were removed during the gel filtration (delipidation) and 10-25 phospholipids were left on a protomer (αβ) of the enzyme irrespective of the cation present during the gel filtration. With the addition of exogenous phospholipids, the activity was restored. The activity of the enzyme delipidated in the presence of KCl was restored to 3-4 times higher than in the case of that delipidated in the presence of NaCl. 3) Relipidation experiments with a fluorescent phospholipid, dansyl phosphatidylethanolamine (Dans-PE), suggested that the enzyme delipidated in the presence of KCl reassociated with phospholipids more firmly than the enzyme delipidated in the presence of NaCl. From these results we concluded that K+ stabilized the (Na, K)-ATPase more effectively than Na+, even when the enzyme was delipidated.View full abstractDownload PDF (584K)
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