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Volume 116 (1994)
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- Issue 5 Pages 939-
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- Issue 3 Pages 471-
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Volume 116, Issue 5
Displaying 1-40 of 40 articles from this issue
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Yohei Miyagi, Naohiko Koshikawa, Hidetaro Yasumitsu, Etsuko Miyagi, Fu ...1994 Volume 116 Issue 5 Pages 939-942
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA serine proteinase inhibitor was purified from conditioned medium of the human glioblastoma cell line T98G. Analysis of its partial amino acid sequences indicated that this protein was identical to placental protein 5 (PP5), a placenta-derived glycoprotein with serine proteinase inhibitor activity, the amino acid sequence of which had been partially determined. cDNA cloning of PP5 demonstrated that it belonged to the Kunitz-type serine proteinase inhibitor family, having three putative Kunitz-type inhibitor domains, and that it was identical to a recently reported inhibitor, tissue factor pathway inhibitor-2 (TFPI-2) [Sprecher et al. (1994) Proc. Natl. Acad. Sci. USA 91, 3353-3357]. PP5/TFPI-2 transcripts were highly abundant in the full-term placenta and widely expressed in various adult human tissues, such as the liver, skeletal muscle, heart, kidney, and pancreas. Several ovarian carcinoma cells as well as T98G also contained significant amounts of the transcriptsView full abstractDownload PDF (3090K) -
Ana Kitazono, Kiyoshi Ito, Tadashi Yoshimoto1994 Volume 116 Issue 5 Pages 943-945
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProlyl aminopeptidase (PAP) has been classified as a sulfhydryl enzyme on the basis of its high sensitivity to p-chloromercuribenzoate and heavy metals. Recently, however, the possibility of PAP being instead a serine enzyme has been raised as a result of two observations-the conservation of some residues among the PAPs hitherto sequenced, and a similarity to some serine hydrolases. This is the first report describing an attempt to identify the active residue by site-directed mutagenesis. The pap genes from Bacillus coagulans and Aeromonas sobria, were used for the study. The changes made were Cys62Ser and Ser101Ala for the first enzyme, and Thr92Ala and Ser146Ala for the second. For both enzymes, only the changes made on the serine residues resulted in their complete inactivation, indicating that PAP is a serine peptidase.View full abstractDownload PDF (382K) -
Chikahiro Nagata, Hideaki Moriyama, Taketomo Fujiwara, Yoshihiro Fukum ...1994 Volume 116 Issue 5 Pages 946-947
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome c-552 from Nitrosomonas europaea was crystallized by the sandwiched drop, vapor diffusion method, with anmmonium sulfate as the precipitant. The crystals were found to belong to the space group P21, 21, 21, having unit cell dimensions of a=106.1 Å, b=126.1 Å, and c=57.7 Å. The crystals diffracted X-rays at greater than 3.0 Å resolution.View full abstractDownload PDF (1798K) -
Shinobu Watarai, Katsuyuki Kiura, Rie Shigeto, Takuo Shibayama, Ikuro ...1994 Volume 116 Issue 5 Pages 948-954
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree kinds of anti-GM1 monoclonal antibodies, AGM-1, -2, and -3, of the IgM class were produced by the immunization of BALB/c mice with ganglioside GM1 inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides and fusion of the spleen cells with a mouse myeloma cell line. The specificities of the monoclonal antibodies obtained were elucidated through complement-dependent liposome immune lysis assay and enzyme immunostaining on thin-layer chromatograms. All of the monoclonal antibodies reacted only with ganglioside GM1, and structurally related glycosphingolipids, such as fucosyl-GM1, asialo-GM1, GM2, and GD1b, and the other gangliosides (GM3 and GD1a) tested showed no reactivity to the 3 monoclonal antibodies. These findings suggest that the monoclonal antibodies obtained may be specific for ganglioside GM1. These anti-GM1 monoclonal antibodies were used to define the expression of ganglioside GM1 on small cell lung carcinoma (SCLC) cell lines and tissues. In flow cytometric analysis and immunostaining studies, we observed that ganglioside GM1 was highly expressed on the SCLC cell lines. Results obtained with flow cytometry and immunohistochemistry agreed well with the immunochemical determination of ganglioside GM1 in lipid extracts of cell lines. Furthermore, expression of ganglioside GM1 in tumor tissues from patients with SCLC was ascertained by the immunohistochemical examination of acetone-fixed paraffin-embedded tissue sections. Ganglioside GM1 was detected in 5 of 19 SCLC tissues. These results suggest that ganglioside GM1 is expressed in SCLC cells.View full abstractDownload PDF (3347K) -
Makiko Takechi, Masayuki Kanda, Kazuko Hori, Toshitsugu Kurotsu, Yoshi ...1994 Volume 116 Issue 5 Pages 955-959
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn gramicidin S-producing Bacillus brevis, the addition of L-ornithine to the minimal medium with L-glutamate as the sole carbon and nitrogen source caused an 8-fold induction of L-ornithine δ-aminotransferase [EC 2. 6. 1. 13]. The enzyme was purified to homogeneity. The native enzyme had a molecular weight of about 88, 000 after gel filtration and consisted of two subunits with an identical in molecular weight of about 45, 000. The enzyme was specific for L-ornithine (Km=1.05mM) as an amino donor and for 2-oxoglutarate (Km=6.25mM) as an amino acceptor, and catalyzed the conversion of L-ornithine and 2-oxoglutarate, respectively, to glutamic-γ-semialdehyde, which is spontaneously cyclized to Δ1-pyrroline-5-carboxylate and L-glutamate. The enzyme exhibits an absorption maximum at 425 nm at neutral pH, and 1 mol of pyridoxal phosphate is bound per subunit. The enzyme activity was irreversibly inhibited by gabaculine, and L-ornithine protected the enzyme from the inhibition. The N-terminal amino acid sequence revealed a noteworthy similarity between human and yeast L-ornithine δ-aminotransferases in residues 17-28 of the B. brevis enzyme.View full abstractDownload PDF (607K) -
Reika Watanabe, Ryoji Masui, Tsutomu Mikawa, Satoko Takamatsu, Ryuichi ...1994 Volume 116 Issue 5 Pages 960-966
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteractions of Escherichia coli RecA protein with ATP and its analogues in the absence of DNA were studied by circular dichroic (CD) spectroscopy. The binding of RecA protein to ATP increased the CD band of ATP at around 260 nm. The positive CD band of the RecA protein-ATP complex suggested that the bound ATP was in the anti conformation, in accord with X-ray crystallographic data [Story, R. M. and Steitz, T. A. (1992) Nature 355, 374-376]. At pH 7.5 and at 25°C the dissociation constant (Kd) and thermodynamic parameters for the binding of ATP to RecA protein were 18 μM (ΔG=-6.5 kcal•mol-1), ΔH=0 kcal•mol-1, and ΔS=22 cal•mol-1-•K-1. A non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thiotriphosphate) (ATPγS), gave a spectral change similar to that of ATP. The Kd for this analogue, 22 μM, was very close to the Km of ATP. These results in the absence of single-stranded DNA were different from those obtained by kinetic analysis [Weinstock, G. M. et al. (1981) J. Biol. Chem. 256, 8850-8855], which indicated that the inhibition constant of ATPγS was much smaller than the Km of ATP in the presence of DNA. For other ATP analogues (dATP, ADP, and dADP), similar spectral changes were observed, and their Kd values ranged from 19 to 54 μM. UTP, dUTP, and TTP also gave CD spectral changes, but not AMP, GTP, dGTP, CTP, and dCTP. The orders of affinity (1/Kd) were: ATP_??_ATPγS_??_ADP_??_AMP for the number of phosphate moieties of the nucleotide, ribose>deoxyribose for the sugar moiety, and A_??_U_??_T_??_G and C for the base moiety.View full abstractDownload PDF (828K) -
Yukio Fujino, Tomoko Fujii1994 Volume 116 Issue 5 Pages 967-972
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResponse to nicotine of adrenal chromaffin cells was studied in suckling and young adult male rats in vivo. When 5mg/kg of nicotine was injected subcutaneously to 8-week-old rats, the content of adrenaline and noradrenaline in the chromaffin granule fraction decreased about by 36 and 45%, respectively, 2min after the administration. In electron microscopy, the number of chromaffin granules in the perinuclear region of adrenaline-storing cells decreased markedly. The number of vacuoles, probably produced by membrane recycling resulting from exocytosis, increased significantly in adrenaline- and noradrenaline-storing cells. Ω-shaped profiles (exocytosis) were frequently observed both in adrenaline- and noradrenaline-storing cells. On the other hand, nicotine injection did not significantly alter the catecholamine content in the 21-day-old rat chromaflin granule fraction, although severe convulsion was evoked. In electron microscopy, the changes indicative of exocytosis mentioned above were scarcely observed. Cholinergic nerve fibers of mature appearance were observed in the adrenal medulla of 21-day-old rats. These results indicate that the responsiveness of the chromaffin cells to nicotine in 21-day-old rats differs from that in 8-week-old rats.View full abstractDownload PDF (5460K) -
Santa J. Tumminia, Paul Russell1994 Volume 116 Issue 5 Pages 973-979
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSαB-crystallin, a major lens protein, is present in non-lenticular tissues. Although αB-crystallin possesses chaperone-like activity, its physiological significance outside the lens is unclear. Recent studies suggest that stress-induced mechanisms of αB-crystallin accumulation are dependent upon the type of insult. To further investigate this possibility, the response of αB-crystallin to different stress conditions was examined in the human astroglioma U373MG cell line which constitutively expresses αB-crystallin. Exposure of this cell line to 25 μM cobalt chloride and heat stress resulted in increased accumulation of αB-crystallin. Exposure to cobalt chloride resulted in a 6-fold (water-soluble) and 5-fold (water-insoluble) increase in αB-crystallin, 4 h after the initial exposure to cobalt. The water-soluble fraction of U373 cells subjected to heat shock at 42°C for 30min and harvested after 1 h exhibited a 4.5-fold increase in the level of αB-crystallin over the control levels.
Approximately 2 h after the removal of the cells from the heat stress, the level of αB-crystallin in the urea-soluble fraction increased 3-fold. Although αB-crystallin accumulation initially increased in response to both the cobalt and heat shock challenges, analysis of the pattern of protein accumulation indicates that there are differences in the mechanism of αB-crystallin induction which are dependent on the type of stress. The concentration of αB-crystallin in both the water-soluble and water-insoluble fractions of heat-stressed cells showed that αB-crystallin concentrations returned to basal levels within 4 h after the initial shock, while levels of αB-crystallin remained high in the water-soluble fraction of cobalt-stressed cells 72 h after the initial stress. Since the role of phosphorylation in chaperone function is controversial, U373 cells were examined for induced forms of phosphorylated αB-crystallin. Isoelectric focusing gel electrophoresis followed by Western analysis indicated that the predominant form of αB-crystallin was unphosphorylated although small amounts of the phosphorylated form were found in all conditions. The phosphorylation state of αB-crystallin did not change after exposure to stress conditions indicating that the protective role of αB-crystallin is phosphorylation-independent under these conditions.View full abstractDownload PDF (4537K) -
Norihiko Misawa, Mark R. Truesdale, Gerhard Sandmann, Paul D. Fraser, ...1994 Volume 116 Issue 5 Pages 980-985
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFull length and truncated cDNA expression constructs of the phytoene synthase (psy) gene from tomato have been ligated into a pUC8 cloning vector. One of the truncated constructs was introduced into Escherichia coli carrying the Erwinia uredovora GGPP synthase gene. This transformant produced 15, 15'-cis-phytoene, which was identified on the basis of its UV and IR spectral data, from geranylgeranyl diphosphate. The function of this gene product was further confirmed by in vitro assay using cell-free extract of E. coli harboring the construct. On transformation with the above constructs together with a plasmid containing the carotenoid gene cluster from E. uredovora devoid of the phytoene synthase (crtB) gene, yellow, carotenoid-containing, E. coli colonies were produced. The amounts of carotenoids synthesized by the transformed cells, related to the steady-state levels of psy mRNA, varied depending upon the psy constructs. The full-length psy clone produced 16-fold less carotenoids per unit amount of RNA than cells containing phytoene synthase without the first 114 N-terminal amino acids. Removal of further amino acids from the N-terminus caused a large decrease in carotenogenesis. A Western blot of ripe fruit stroma with a monoclonal antibody raised against phytoene synthase revealed a single protein band of apparent molecular mass 38 kDa. Based upon this immunological evidence, we conclude that the size of the transit peptide of phytoene synthase from ripe tomato fruit is approximately 9 kDa, corresponding to about 80 amino acid residues. But removal of further N-terminal amino acids up to 114 from the enzyme, to yield a protein of apparent molecular mass 34 kDa, increases its catalytic activity in E. coli.View full abstractDownload PDF (1983K) -
Ya-Ming Ger, Shih-Lin Chen, Huey-Jenn Chiang, David Shiuan1994 Volume 116 Issue 5 Pages 986-990
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Escherichia coli phenylalanine-sensitive DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate) synthetase (aroG product) is one of the DAHP synthetase isozymes that catalyze the first committed step in the biosynthesis of aromatic amino acids and vitamins. Through target-directed mutagenesis of the cloned aroG on a plasmid vector, followed by screening of phenylalanine-resistant colonies, we isolated a clone (pG908) showing feedback-insensitive mutation of DAHP synthetase. The mutations were identified as a T→A mutation at nucleotide 22, and a C→T mutation at nucleotide 539, causing a Leu-8 to Ile-8 mutation and a Ser-180 to Phe-180 substitution, respectively. The resulting enzyme exhibited comparable enzymatic activity to the wild type, but the degree of feedback inhibition had declined from approximately 60% to less than 10% in the presence of 20mM phenylalanine in the assay medium. Replacement of Ile-8 by Leu, and substitution of Phe-180 with Ser, Asn and Cys, using site-directed mutagenesis, demonstrated that Ser-180 is a critical residue in the feedback inhibition of AroG. The result suggests that the major role played by Ser-180 may not involve a simple charge-size effect in the Phe-binding site of the enzyme molecule, but rather may involve more complicated molecular interactions occurring in the feedback inhibition mechanisms.View full abstractDownload PDF (651K) -
Yoshimitsu Kimura, Kazuhiko Sonehara, Etsuro Kuramoto, Tadashi Makino, ...1994 Volume 116 Issue 5 Pages 991-994
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpecific palindromic sequences in synthetic oligonucleotides are required to induce IFN and augment IFN-mediated natural killer activity. To study the mechanism of IFN induction by oligonucleotides containing palindromic sequences, we investigated the possible target molecules of the oligonucleotides. Oligo-1, a 30 mer single-stranded oligonu-cleotide with oligoG sequences next to the active palindromic sequence (AACGTT), had more activity than oligonucleotides with oligoA, oligoC, or oligoT sequences. The activity of oligo-1 was inhibited by a guanine homo-oligomer (G30), dextran sulfate, and polyvinyl sulfate. Oligo-1 bound to plastic-adherent mouse splenocytes, and the binding was inhibited by G30, dextran sulfate, and polyvinyl sulfate. Oligo-1 inhibited acetyl-LDL binding to the scavenger receptor on mouse splenocytes. These findings suggest that the binding of an extrapalindromic sequence to the scavenger receptor is required for the immunostimu-latory activity of oligo-1.View full abstractDownload PDF (1700K) -
Yoshihiro Sawa, Masaaki Tani, Ken Murata, Hitoshi Shibata, Hideo Ochia ...1994 Volume 116 Issue 5 Pages 995-1000
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240 kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH2 terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD+, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The Km, values were 5.0mM for L-alanine and 0.04mM for NAD+, 0.33mM for pyruvate, 60.6mM for NH4+ (pH 8.7), and 0.02mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP.View full abstractDownload PDF (765K) -
Ikuko Miyahara, Ken Hirotsu, Hideyuki Hayashi, Hiroyuki Kagamiyama1994 Volume 116 Issue 5 Pages 1001-1012
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe three-dimensional structures of pyridoxamine 5'-phosphate-type aspartate amino-transferase from Escherichia coli and its complexes with maleate and glutarate have been determined by X-ray crystallography at 2.2, 2.1, and 2.7 Å resolution, respectively. The enzyme is a dimeric form comprising two identical subunits, each of which is divided into one large and one small domain. The complex with maleate showed that substrate (or inhibitor) binding induced a large conformational change from the “open” to the “closed” form, resulting in closure of the active site by the small domain movement, as was observed in the pyridoxal 5'-phosphate-type enzyme. In the open form, three hydrophobic residues (hydrophobic plug) at the entrance of the active site are exposed to solvent. Maleate binding make the active site more hydrophobic by charge compensation and release of water molecules, facilitating the movement of the hydrophobic plug into the active site pocket to induce a large conformational change in the enzyme. Maleate is fixed rigidly in the active site pocket by extensive salt bridges and a hydrogen bonding network, guaranteeing the stereo-specificity of the catalysis and giving a Michaelis complex model. Contrary to our expectation, the glutarate complex was in the open form, suggesting that the equilibrium between the open and closed forms lies far toward the open form in solution. The water molecules located in the active site pocket were almost completely conserved between Escherichia coli and chicken mitochondrial aspartate aminotransferase with the same type of cofactor and the same conformation.View full abstractDownload PDF (1446K) -
Kaeko Hayashi, Tohru Takehisa, Nobuaki Hamato, Ryo Takano, Saburo Hara ...1994 Volume 116 Issue 5 Pages 1013-1018
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSquash family inhibitors are the smallest protein serine protease inhibitors, being composed of approximately 30 amino acid residues. We isolated 8 squash family inhibitors from the seeds of bitter gourd, squash, gourd and luffa and examined their effect on serine proteases of the blood coagulation system. Five of them prolonged the activated partial thromboplastin time of human plasma to various extents, but three did not. Only Momordica charantia (bitter gourd) trypsin inhibitor-II prolonged the prothrombin time of human plasma. All inhibitors inhibited the amidolytic activities of factor XIIa, plasma kallikrein, factor Xa, but did not inhibit significantly those of factor XIa, factor IXa, factor VIIa, and thrombin. Ki values for factor XIIa, plasma kallikrein, and factor Xa were in the order of 10-6-10-9, 10-4-10-5, and 10-4-10-6M, respectively. The prolongation of the activated partial thromboplastin time by inhibitors appeared to correspond to their inhibitory potencies for factor XIIa. Momordica charantia trypsin inhibitor-II, which has the strongest inhibitory potency toward the amidolytic activity of factor Xa, with a Ki value 10-100 times smaller than those of other inhibitors, inhibited the activation of factor X by factor VIIa-tissue factor complex or factor IXa, while others did not.View full abstractDownload PDF (809K) -
Yoshizumi Ishino, Takashi Ueno, Masaru Miyagi, Takashi Uemori, Mitsuo ...1994 Volume 116 Issue 5 Pages 1019-1024
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe cloned the pol gene from the Thermus aquaticus YT-1 strain into a plasmid vector and constructed a high-level expression system of the gene in Escherichia coli. Six codons in the translational start region were changed to simple AT-type codons or codons which are most frequently used in E. coli by the genetic engineering techniques with retention of the amino acid sequence of the native enzyme. The modified pol genes were expressed under the lac promoter of pUC-type plasmid and 266, 418 units of activity was obtained in a sonicated and heated crude extract from 2g of E. coli cells bearing one of the recombinant plasmids, pTAQ9. Highly purified protein was subjected to structural analysis using a protein sequencer and an ion-spray mass spectrometer combined with reversed-phase HPLC (LC-MS). The primary structure of the DNA polymerase was identical with the amino acid sequence deduced from the nucleotide sequence of the pol gene as far as examined (about 95% of the sequence); though, several regions where small peptides of less than 5 residues were produced by lysyl endopeptidase digestion could not be sequenced.View full abstractDownload PDF (2692K) -
Yoko Honma, Manabu Niimi, Toshio Uchiumi, Yoshiaki Takahashi, Shoji Od ...1994 Volume 116 Issue 5 Pages 1025-1029
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConformational change of rat liver fatty acid-binding protein was investigated by making use of change of protease-susceptibility in the presence or absence of bound oleic acid and clofibrate. Delipidated fatty acid-binding protein was rapidly digested by Achromobacter lysyl endopeptidase, bovine α-chymotrypsin, and Staphylococcal V8 protease, while protein recombined with oleic acid was strongly refractory to proteolysis. This observation indicates that a striking change in the conformational state of the protein occurred upon lipid binding, and seems to support the recent hypothesis that the large segment homologous to fatty acid-binding proteins found in a fatty acid-regulated ion channel constitutes a regulatory domain of the channel [Petrou, S., Ordway, R. W., Singer, J. J., and Walsh, J. V., Jr. (1993) Trends Biochem. Sci. 18, 41-42]. Clofibrate, which is a potent peroxisome proliferator and structurally unrelated to oleic acid, also conferred similar protease resistance upon the protein. A possible physiological aspect of the present observation is that the cellular level of free fatty acids, which have metabolic importance and cytotoxicity as well, regulates the turnover rate of fatty acid-binding proteins by modulating the protease susceptibility of the protein.View full abstractDownload PDF (575K) -
Teruo Kuroda, Toshi Shimamoto, Kei Inaba, Masaaki Tsuda, Tomofusa Tsuc ...1994 Volume 116 Issue 5 Pages 1030-1038
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of the slightly halophilic marine bacterium Vibrio parahaemolyticus. The host was an Escherichia coli mutant that lacked both of the two major Na+/H+ antiporters, NhaA and NhaB. Untransformed mutant cells were unable to grow in the presence of 0.6 M NaCl or 0.1 M LiCl, but Na+ and Li+ were non-toxic to cells transformed with a plasmid carrying the antiporter gene. Membrane vesicles prepared from the original E. coli mutant did not show any detectable Na+/H+ (and Li+/H+) antiport activity. However, we observed high Na+/H+ (and Li+/H+) antiport activity in membrane vesicles prepared from the transformed cells. The activity increased greatly when the pH of the assay medium was increased from 7.0 and 8.5. This property is very similar to that of the NhaA Na+/H+ antiporter of E. coli. Drastic decreases in Km values for Li+ and Na+ were observed with membrane vesicles prepared from the transformed cells compared with those observed with V. parahaemolyticus vesicles. The amino acid sequence deduced from the nucleotide sequence of the cloned gene showed high homology (59% identity and 87% similarity) with the NhaA Na+/H+ antiporter of E. coli. Thus, we conclude that the gene we cloned and sequenced is the nhaA of V. parahaemolyticus. We also found that several regions of the NhaA protein showed sequence similarity with transport proteins from some other organisms. Such regions seem to be important for Na+ recognition, transport or amiloride binding.View full abstractDownload PDF (1906K) -
Ichiro Tokimitsu, Makoto Takehana, Hisae Hori, Yutaka Nagai, Shingo Ta ...1994 Volume 116 Issue 5 Pages 1039-1043
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo collagenous polypeptides with apparent molecular weights of 180 and 500 kDa under a nonreducing condition were found to be produced in the culture medium of B16 mouse melanoma cells. Under a reducing condition, the higher molecular weight polypeptide (500 kDa) migrated at an identical position to the 180 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both polypeptides were found to have an identical primary structure by V8 protease peptide mapping. Cyanogen bromide cleavage patterns, the fragments produced by pepsin digestion, and partial amino acid sequences of the 180 kDa polypeptide were compatible with those of proα1(IV) chain. These results indicate that B16 mouse melanoma cells preferentially produce prowα1(IV) chains consisting of two forms: disulfide- and nondisulfide-bonded forms.View full abstractDownload PDF (2923K) -
Tadashi Matsubasa, Masaki Takiguchi, Ichiro Matsuda, Masataka Mori1994 Volume 116 Issue 5 Pages 1044-1055
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene for rat argininosuccinate lyase (AL), which catalyzes the last step of arginine biosynthesis, is expressed in many tissues, though its expression is much higher in the liver where the enzyme is involved in the ornithine cycle (urea cycle), and moderately higher in the kidney. In transient transfection analysis using various cell lines, the AL promoter exhibited considerable levels of activity in all tested cell lines, with no apparent liver cell specificity, presumably reflecting the ubiquitous expression of this gene. Analysis of 5'-deletion mutants of the promoter region revealed several cis-regulatory elements, whose contribution to the promoter activity seemed to vary according to cell type. The most prominent positive regulatory region was detected around the position -80 base pairs, and it overlapped with a dyad-symmetric CCAAT box sequence CCAATTGG. A factor(s) interacting with this sequence element was found to be present ubiquitously. Gel shift competition analysis and antibody double shift analysis showed that this factor was identical or highly similar to a transcription factor, nuclear factor-Y (NF-Y), which consists of two different subunits and recognizes CCAAT motifs in various promoters. Point mutagenesis at each half site of the palindrome CCAATTGG and the introduction of a space between the half-site motifs abolished the binding with NF-Y. Thus, it seems that NF-Y recognizes the correctly spaced dyad-symmetric CCAAT box.View full abstractDownload PDF (7072K) -
Tuomo A. Keinänen, Tapani Hyvönen, Marvin C. Pankaskie, Jouk ...1994 Volume 116 Issue 5 Pages 1056-1062
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1-or 3-methylated derivatives and oximes of 1-aminooxy-3-aminopropane (APA) with pyridoxal (PL) and pyridoxal 5'-phosphate (PLP) were synthesized to examine whether the stability of the parent APA molecule could be increased without loss of its inhibitory capacity towards ornithine decarboxylase. Preformed APA-PLP was more stable than APA and was not a substrate of cellular acetylating activity. The only detectable degradation mechanism of APA-PLP was a slow dephosphorylation to APA-PL, which was a substrate for cellular acetylating activity like the methylated APA derivatives. Methylation at the 1 or 3 position of APA did not increase its stability but markedly changed its inhibitory potency towards S-adenosylmethionine decarboxylase and spermidine synthase. Supplementation of cell growth media with 1mM aminoguanidine markedly reduced the degradation rate of 1- or 3-Me-APA and APA. All the growth-retarding effects of the drugs were reversed by addition of 10-20 μM putrescine or spermidine to the growth media containing a drug concentration of 1mM, except with APA-PL, which had signs of emergent toxicity at concentrations above 0.5mM. APA-PL and APA-PLP were as good as APA and two orders of magnitude more effective than α-difluoromethylornithine (DFMO) in inhibiting DNA synthesis by BHK21/C13 cells.View full abstractDownload PDF (953K) -
Mitsuhiko Take, Jun-ichiro Tsutsui, Hiroya Obama, Masayuki Ozawa, Tats ...1994 Volume 116 Issue 5 Pages 1063-1068
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa, and is structurally unrelated to fibroblast growth factors (FGF). We studied MK-binding proteins in order to clarify the action mechanism of MK, A 100-kDa protein was identified in PYS-2, 3T3, and L cells as an MK-binding protein by a ligand blot experiment. This MK-binding protein was purified by affinity chromatography on an MK-agarose column followed by SDS polyacrylamide gel electrophoresis. Sequence determination of N-terminal 23 amino acid residues revealed that the MK-binding protein was nucleolin, a major nucleolar protein, which functions as a shuttle protein between the nucleus and cytoplasm and is located also on the cell surface. Heparin-binding growth associated molecule (HB-GAM), which has 50% sequence identity with MK, fused to maltose-binding protein also bound to nucleolin. On the other hand, basic FGF (bFGF) scarcely bound to nucleolin in the absence of heparin, while both MK and bFGF bound weakly to nucleolin in the presence of heparin. Nuclear localization of MK was shown in hemangioma cells by immunohistochemical staining. These findings supported the hypothesis that parts of the MK and HB-GAM are translocated to the nucleus after binding with nucleolin.View full abstractDownload PDF (2936K) -
Shinji Asano, Motoya Ohshika, Noriaki Takeguchi1994 Volume 116 Issue 5 Pages 1069-1074
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMonoclonal antibody HK4013 raised against hog gastric vesicles dose-dependently inhibited gastric H+, K+-ATPase activity, formation of phosphoenzyme from ATP, and proton uptake into gastric vesicles. This antibody did not cross-react with related P-type ATPases such as hog kidney Na+, K+-ATPase or rabbit sarcoplasmic reticulum Ca2+-ATPase. It did not bind to the solubilized gastric H+, K+-ATPase, indicating that this antibody recognizes a higher-order structural epitope. The epitope is present on the cytosolic surface of H+, K+-ATPase. The addition of K+ to a solution containing gastric H+, K+-ATPase decreased the fluorescence intensity of the enzyme labeled with fluorescein isothiocyanate (FITC), showing the conformational transition from the E1 to E2K+ form. When H+, K+-ATPase was preincubated with HK4013, the addition of K+ did not decrease but increased the FITC fluorescence, indicating that this antibody changed the conformational state, at least near the ATP binding site. This is in contrast with the case of monoclonal antibody HK4001, which inhibited the decrease of the fluorescence. The fact that these two antibodies recognize different epitopes is consistent with previously reported facts that monoclonal antibody HK4001 inhibits the ouabain-insensitive K+-ATPase activity of rat distal colon but antibody HK4013 does not, and that the former stains the rabbit distal H+, K+-ATPase but the latter does not.View full abstractDownload PDF (756K) -
Mizue Morioka, Hiromichi Muraoka, Kazuo Yamamoto, Hajime Ishikawa1994 Volume 116 Issue 5 Pages 1075-1081
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSymbionin, a GroEL homologous molecular chaperone produced by an intracellular symbiont of the pea aphid, is able to transfer its high-energy phosphate bond to other compounds through its autophosphorylation. When the urea-dissociated monomeric symbionin fixed onto a polyvinylidene difluoride membrane was incubated with [γ-32P] ATP, it was efficiently phosphorylated at elevated temperatures. The autophosphorylated monomeric 32P-labeled symbionin, when incubated with ADP, transferred a significant portion of its radioactivity to ADP, suggesting that the autocatalytically phosphorylated monomeric symbionin contains high-energy phosphate bonds. It was also shown that when symbiotic proteins were electrophoretically separated, blotted onto a polyvinylidene disulfide membrane and incubated with 32P-labeled symbionin, radioactivity was found on several kinds of polypeptides, indicating that the phosphoryl group was transferred from symbionin to other symbiotic proteins. Peptide sequence analysis and thin-layer chromatographic analysis of the 32P-labeled tryptic fragment of the phosphorylated symbionin revealed that the site of phosphorylation is His-133. These results suggested that symbionin functions as a histidine protein kinase, or a sensor molecule, of the two-component pathway known in other organisms. However, symbionin is not similar in amino acid sequence to any known histidine protein kinase.View full abstractDownload PDF (5060K) -
Masahiro Nishijima, Sayuri Hara-Kuge, Naomi Takasuka, Kiyoko Akagawa, ...1994 Volume 116 Issue 5 Pages 1082-1087
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously isolated a lipopolysaccharide (LPS)-resistant mutant (named LR-9) of a cultured macrophage-like cell line, J774.1. This mutant had defective LPS binding [Hara-Kuge, S., Amano, F., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 6606-6610]. In this study, we found that: (1) LPS-binding to parental J774.1 cells was dependent on a serum factor with a molecular weight of about 60 kDa, probably LPS binding protein (LBP); (2) LPS-binding to J774.1 cells was markedly reduced by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC); (3) mutant LR-9 cells were defective in LPS-binding even in the presence of serum; (4) LR-9 cells lacked CD14 protein on flow cytometric and immunoblot analyses, but retained normal CD14 mRNA levels on RNA blot analysis; (5) small amounts of LPS (1 to 10 ng/ml) activated J774.1, but not LR-9 cells, to secrete tumor necrosis factor-α and to release arachidonate metabolites, whereas both J774.1 and LR-9 were activated by large concentrations of LPS (100 to 1, 000 ng/ml). These results provide genetic evidence that CD14 molecules in J774.1 cells play a crucial role in LPS-binding and in LPS-triggered signal transduction, and indicate that large amounts of LPS can activate J774.1 cells without the participation of CD14 molecules.View full abstractDownload PDF (2601K) -
Elena Bravo, Kathleen M. Botham, Malcolm A. Mindham, Peter A. Mayes, T ...1994 Volume 116 Issue 5 Pages 1088-1095
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs part of a study of the effects of aging on lipoprotein metabolism, the uptake and processing in vivo of cholesterol from high density lipoprotein (HDL) was compared in young (3 months of age) and mature (10-12 months of age) rats by studying the fate of HDL [3H] unesterified cholesterol or [3H] cholesteryl ester after intravenous administration. Radioactivity from [3H] unesterified cholesterol was cleared from the blood more slowly in older rats, and this difference was accounted for by decreased uptake by the liver. Uptake by other tissues was unaffected. In addition, a shift in the distribution of radioactivity across the plasma lipoprotein density range from the d=1.125-1.250g/ml (HDL3) to the d=1.050-1.085g/ml (HDL1) fraction was observed in the mature as compared to the young rat group. The secretion of radioactivity from [3H] unesterified cholesterol into bile was also decreased in the older animals, particularly in the first hour after injection of the label. In the case of HDL labeled with [3H] cholesteryl ester, clearance from the blood was similar in both age groups in the first 30min after injection, but was significantly lower in older rats at later time points. After 180min, less radioactivity was found in the VLDL density fraction in mature as compared to young rats, suggesting that hepatic secretion of VLDL cholesterol originating from HDL cholesteryl ester is less efficient in the older animals. The amount of radioactivity from HDL [3H] cholesteryl ester secreted in bile was less in the mature rat group at all time points measured. In addition, labeled bile acid from both HDL unesterified and esterified cholesterol accumulated in the liver to a greater extent in older as compared to younger animals. These results demonstrate that the efficiency of reverse cholesterol transport associated with HDL decreases with age in the rat, and that both slower biliary excretion and slower hepatic metabolism of HDL unesterified cholesterol and cholesteryl ester contribute to this process. It is likely that this effect is associated with the increase in plasma cholesterol levels which occurs with age. Caution is necessary when applying these results to species having the cholesteryl ester transfer protein.View full abstractDownload PDF (1105K) -
Manabu Gomada, Hiromasa Imaishi, Koshiro Miura, Sachiye Inouye, Teruko ...1994 Volume 116 Issue 5 Pages 1096-1104
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe transcription of both the upper operon (OP1) coding for m-xylene-degrading enzymes and the positive regulatory gene xylS on the TOL plasmid depends on σ54-RNA polymerase and requires the activator protein XylR that binds to the cis-acting upstream regulatory sequence of each promoter. For transcription of OP1 in Escherichia coli, integration host factor (IHF) is also required. IHF binds to DNA between the upstream regulatory sequence and the promoter sequence of OP1. We showed that IHF induced a DNA bend in the promoter regulatory region of OP1 upon its binding, supporting the DNA-loop model for the activation of OP1 transcription. In contrast to OP1, the transcriptional activation of xylS does not require IHF. In the absence of IHF, the promoter regulatory region of xylS promoter was shown to have a weak but significant intrinsic DNA bend, which may be involved in stabilizing the DNA-loop structure for the activation of xylS transcription. When IHF was highly produced in E. coli, the xylS transcription was repressed. We found two weak binding sites for IHF, each overlapping with the promoter sequence and the upstream regulatory sequence. The IHF binding to these sites might result in repression of xylS expression by overproduced IHF. Evidence is presented that binding of IHF to these sites induces a DNA bend.View full abstractDownload PDF (6081K) -
Masanori Terajima, Yasuhisa Matsui, Neal G. Copeland, Debra J. Gilbert ...1994 Volume 116 Issue 5 Pages 1105-1110
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe human glycophorin gene has been extensively studied, but information on the homologous gene from other species has been unavailable. Here, we determined the structural organization of mouse glycophorin A gene and compared it with the human gene. The mouse glycophorin gene is a single copy gene while in humans, there are two highly related genes (A and B) that were generated by homologous recombination. Chromosomal mapping indicated that the mouse gene is located in the central region of the mouse chromosome 8, which is syntenic with human chromosomes 4q28-31 where the human glycophorin A gene has been mapped. The mouse gene consists of 8 exons, while the human gene consists of 7 exons and the length of each exon is quite short except for the last exon. The last 4 exons showed extensive homology between the mouse and human genes but divergence in the 5'-exons of the two genes was high. The results suggest that glycophorin genes of mouse and human may have been generated from the same ancestor, but diverged
greatly during evolution. The upstream regulatory region of the mouse gene consists of multiple motifs for DNA binding factors that may be required for its erythroid-specific expression.View full abstractDownload PDF (1685K) -
Tsutomu Mori, Yasuyoshi Oshima, Hideo Kochi1994 Volume 116 Issue 5 Pages 1111-1116
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding characteristics among three catalytic components of rat liver branched-chain 2-oxo acid dehydrogenase complex (BCKADH) were investigated by ELISA. Dihydrolipoamide dehydrogenase (E3) was bound to solid-phase dihydrolipoamide acyltransferase (E2). The binding curve was hyperbolic giving a calculated half-maximal binding concentration of 167 ng/ml for E3. Specificity of the binding of E3 to E2 was certified by a competition experiment measuring binding of biotin-labeled E3 in the presence of unlabeled E3. The decarboxylase component (E1), which is the other catalytic component of the complex, prevented the E3 binding to E2. The specific binding between E2 and E3 was verified in the opposite direction with immobilized E3. E2 also bound to solid-phase E1 in a specific manner, and addition of E3 prevented the E2 binding to E1. No binding between E1 and E3 was observed. Thus, E1 and E3 prevented each other's binding to E2, suggesting that E1 and E3 may recognize overlapped binding sites on the E2 polypeptide or that they may, at least in part, sterically interact with each other on their binding to E2. The reconstitution experiment of the complex also supported such a mutually exclusive binding of E1 and E3 to the E2 core.View full abstractDownload PDF (693K) -
Takashi Murayama, Yasuo Ogawa1994 Volume 116 Issue 5 Pages 1117-1122
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo clarify the relationship among ryanodine receptor isoforms from skeletal muscles of various vertebrates (α- and β-isoforms with smaller and larger mobilities on SDS-poly-acrylamide gel electrophoresis, respectively), we examined the cross-reactivities of those receptors from 13 animal species by Western blot analysis, using specific polyclonal antibodies raised in rabbit against the bullfrog α- and β-isoforms. Anti-β-isoform antibody showed positive reactions against skeletal muscles from carp, five species of frogs, two species of toads, and chicken, but it was negative with four mammals. Positive reactions with anti-α-isoform antibody, by contrast, were limited to four species of frog Rana, although the α-isoforms from toads (Xenopus and Bufo) also reacted very weakly. The β-isoforms from various animal species showed almost the same mobilities on SDS-PAGE in contrast to the variable mobilities of the α-isoforms. These results indicate that the β-isoform in one animal species corresponds to the β-isoform in other species and that the β-isoforms are highly conserved. It is also suggested that the α-isoforms may be related to each other, although they are more diversified.View full abstractDownload PDF (4184K) -
Nobuo Yamaga, Hiromi Kohara1994 Volume 116 Issue 5 Pages 1123-1126
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBile acids extracted from the urine of a healthy volunteer who excreted 7β-hydroxylated bile acids were fractionated to nonamidated, glycine-conjugated, taurine-conjugated, and sulfated bile acid fractions. The chemical conjugation types of the 7β-hydroxylated bile acids were then determined by treatment with several enzymes and by capillary column gas chromatography. Large amounts of 3α, 7β, 12α-trihydroxycholanoic acid were present as nonamidated and nonconjugated bile acids, while 3β, 7β-dihydroxycholanoic acid formed nonamidated bile acid N-acetylglucosaminide. In addition, ursodeoxycholic acid formed both glycine-conjugated bile acid and glycine-conjugated bile acid N-acetylglucosaminide. Bile acid N-acetylglucosaminides were hydrolyzed by solvolysis.View full abstractDownload PDF (471K) -
Taei Matsui, Yasuhiro Ozeki, Masami Suzuki, Akiya Hino, Koiti Titani1994 Volume 116 Issue 5 Pages 1127-1133
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo structurally distinct lectins were purified from the coelomic plasma of holothurian, Stichopus japonicus, by affinity chromatography on a porcine stomach mucin-conjugated agarose column, gel filtration on a Superose 6 column, and ion-exchange chromatography on a HiTrap Q-FPLC. The two lectins showed apparent molecular masses of about 400 kDa (SPL-1) and 60 kDa (SPL-2) on gel filtration, but about 17 kDa on SDS-PAGE under reducing conditions. Both lectins showed hemagglutination activity toward rabbit erythrocytes in the presence of Ca2+ ions. The N-terminal amino acid sequences were highly homologous to but distinct from those of a Ca2+-dependent (C-type) lectin named SJL-I purified from the same species. In addition to porcine stomach mucin, the hemagglutinatioi activity of SPL-1 was strongly inhibited by uronic acids such as galacturonic acid, and glucuronic acid, while the activity of SPL-2 was inhibited by GalNAc and galactosides. Both lectins were adsorbed on clotted coelomocytes in the presence of Ca2+ but not in the presence of inhibitory sugars or EGTA, suggesting the presence of an endogenous carbohydrate ligand(s) for plasma C-type lectins in the clot. However, coelomocyte clotting occurred normally even in the presence of inhibitory sugars, but was strongly inhibited by synthetic GRGDSP peptide or EGTA, suggesting the participation of integrin but not the lectin-carbohydrate interaction in the clotting events.View full abstractDownload PDF (2864K) -
Tomohiko Maehama, Hiroshi Nishina, Toshiaki Katadat1994 Volume 116 Issue 5 Pages 1134-1138
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA rat glycohydrolase which catalyzes the hydrolysis of ADP-ribosylarginine was expressed in Escherichia coli and purified to homogeneity for characterization of its enzymatic properties. The purified glycohydrolase catalyzed the hydrolysis of N-glycoside linked ADP-ribosylarginine on the α-subunits of stimulatory GTP-binding proteins (Gs) and cholera toxin A1-subunit that had been modified by cholera toxin and NAD. Nonmuscle actin of which an arginine residue was ADP-ribosylated by botulinum C2 toxin also served as a substrate of the glycohydrolase. On the other hand, the glycohydrolase did not hydrolyze ADP-ribosylated cysteine on the α-subunits of pertussis toxin-substrate GTP-binding proteins, ADP-ribosylated diphthamide on elongation factor 2, or ADP-ribosylated asparagine on rho GTP-binding proteins. The rate of the reaction catalyzed by the glycohydrolase was affected by nucleotide-binding form of the ADP-ribosylated substrate proteins; the GDP-bound form of the modified Gs-α was more rapidly hydrolyzed than the guanosine 5'-(3-O-thio) triphosphate-bound form. Interestingly, the glycohydrolase activity was markedly inhibited by mM order concentration of ATP in addition to ADP-ribose, the product of the enzyme reaction, though ADP had no inhibitory effect on the activity. Moreover, αNAD, but not βNAD, inhibited the enzyme activity, suggesting that the glycohydrolase reaction was stereospecific for the α-anomer.View full abstractDownload PDF (3046K) -
Mi-Yeon Park, Hiroshi Omote, Masatomo Maeda, Masamitsu Futai1994 Volume 116 Issue 5 Pages 1139-1145
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwenty-two mutants between βGlu-161 and βLys-201 of Escherichia coli H+-ATPase β subunit could grow by oxidative phosphorylation, but 11 other such mutants, βGlu-181→Gln, Asp, Asn, Thr, Ser, Ala, or Lys and βArg-182→Lys, Ala, Glu, or Gln, could not. The βAsp-181, β3Lys-182, and other defective mutants had 1.4, 1, and <0.1%, respectively, of the wild-type membrane ATPase activity. Partially purified F1-ATPases from all mutants at positions 181 and 182, except for the, βAsp-181 and βLys-182 mutants, showed very low unisite catalysis. Purified F1-ATPases of the β3Gln-181 and βAla-181 mutants showed no multisite (or steady state) catalysis and slow unisite catalysis (≤1% of that of the wild type): their defects could be attributed to decreased catalytic rates (low k+2 and k-2). Changes of the k+2 and k-2 values in the, βAsp-181 enzyme, which showed detectable multi- and unisite catalysis, were less marked (27 and 21%, respectively, of wild-type rates). The β3Gln-182 enzyme showed defective catalysis (≤0.1% of the multi- and _??_1% of the unisite catalyses of the wild type), whereas the βLys-182 enzyme showed 1 and 85% of the wild-type multisite and unisite catalytic rates, respectively. βLys-182 had wild-type values of k+2 and k-2, but βGln-182 had k+2 about 10-fold lower than that of wild type. The position 181 and 182 mutant enzymes had significantly increased Kd (k-1, / k+1) values, reflecting decreased substrate binding. These results suggest that βGlu-181 and βArg-182 are essential for substrate binding, although mutations with conservative substitutions at these positions do not have drastic effects. This study also indicates the importance of the conserved Gly-Glu-Arg (GER) sequence (β180-β182).View full abstractDownload PDF (2101K) -
Futoshi Masuya, Motonari Tsubaki, Ryu Makino, Hiroshi Hori1994 Volume 116 Issue 5 Pages 1146-1152
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhotolyzed products of the NO complexes of ferric cytochrome P450cam both in the substrate-free and several substrate-bound states were trapped and examined by EPR spectroscopy at 5K. In the absence of substrate, the photoproduct exhibited ferric high- and low-spin signals, neither of which showed the line-width broadening characteristic of magnetic interaction between photodissociated NO and the heme iron. This finding indicates that photodissociated NO can diffuse to the unconstrained distal heme pocket giving the high-spin heme, and that a part of the high-spin heme species converts to the low-spin heme upon coordinating an aqua molecule. When a substrate, camphor or adamantanone, was bound at the site above the heme, the photoproduct exhibited widespread EPR absorptions together with a new distinct signal at g_??_4.4. The new signals are assignable to a weakly spin-coupled species between the ferric heme iron and the photodissociated NO, indicating that the NO molecule is in close proximity to the heme iron by the steric crowding of the bound substrate. The photoproduct of the norcamphor complex exhibited a spin-coupled EPR signal at g_??_5, in which the coupling is suggested to be weaker than that of the camphor-bound enzyme. On the other hand, the photoproduct of the NO complex in an adamantane-bound state only yielded low-spin signals, and exhibited no spin-coupled signals. This result suggests that adamantane is mobile in the substrate pocket due to the lack of hydrogen bond formation with Tyr96. Thus, EPR examinations of the photolyzed NO complex at 5K provide information on the constraints of the distal heme cavity. Based on the results, the structure of the heme vicinity in cytochrome P450cam is discussed.View full abstractDownload PDF (978K) -
Yoshimitsu Kakuta, Hironobu Hojo, Saburo Aimoto, Isao Tanaka, Kunio Hi ...1994 Volume 116 Issue 5 Pages 1153-1155
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mobility of the DNA-binding arm of HU protein was studied by 13C-NMR spectroscopy. The correlation times τc of Phe47Cα in the body and Gly60Cα in the arm of HU were determined for HU and HU-DNA complex. The value of τc of Phe47Cα is 2-4 times larger than that of Gly60Cα irrespective of the presence or absence of DNA. These results show that Gly60Cα undergoes more rapid motion than Phe47Cα. The increase in correlation time on addition of DNA is greater for Gly60Cα than for Phe47Cα. This suggests that the addition of DNA influences more significantly the motion of Gly60Cα than that of Phe47Cα. These results are in accord with the X-ray result, in which the top part of the arm is not visible. Gly60Cα in the arm is thus more mobile than Phe47Cα in the body, and the mobility of Gly60Cα is reduced by the DNA binding.View full abstractDownload PDF (324K) -
Mahito Terabe, Shuichi Kojima, Seiichi Taguchi, Haruo Momose, Kin-ichi ...1994 Volume 116 Issue 5 Pages 1156-1163
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree novel proteinaceous inhibitors, which had been identified as “Streptomyces subtilisin inhibitor-like (SIL) proteins” and exhibited trypsin inhibition in addition to strong inhibition toward subtilisin BPN', were purified from the culture broth of three Streptomyces strains: SIL10 from S. thermotolerans, SIL13 from S. galbus, and SIL14 from S. azureus. Their primary structures were determined by sequence analysis of intact SIL inhibitors and peptides obtained by enzymatic digestions of S-pyridylethylated SIL inhibitors. These inhibitors were composed of about 110 amino acids and existed as dimer proteins. The reactive site was identified as Lys-Gln for all three inhibitors by sequence analysis of their modified forms in which the reactive-site peptide bond was specifically cleaved by subtilisin BPN' under acidic conditions. Thus, their inhibition toward trypsin and subtilisin BPN' was due to the presence of a Lys residue at the P1 site. Inhibitor constants toward subtilisin BPN' and trypsin were also determined. These inhibitors showed relatively high sequence homology to other SSI-family inhibitors possessing a Lys residue at the P1 site, with amino acid replacements on their molecular surface.View full abstractDownload PDF (2852K) -
Fuminori Tokunaga, Tamami Goto, Sadao Wakabayashi, Takehiko Koide1994 Volume 116 Issue 5 Pages 1164-1170
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAntithrombin III (ATIII) is a member of the serine protease inhibitor (serpin) family. As a step towards a better understanding of the heparin-binding mechanism of mammalian ATIIIs, the amino acid sequence of porcine ATIII was established by sequence analysis of the peptides derived from cyanogen bromide cleavage and enzymatic digestion with lysyl endopeptidase, V8 protease, and trypsin. Porcine ATIII was found to consist of 431 amino acid residues, with a calculated molecular weight of 48, 930 without carbohydrate. Its molecular weight with 16.4% carbohydrate was estimated as 56, 955, which is in good agreement with the value determined by SDS-PAGE. Porcine ATIII showed high sequence similarity to other mammalian ATIIIs, including the reactive site, heparin-binding basic amino acid residues, and disulfide bonds. The most notable feature of porcine ATIII was that it possesses only three carbohydrate chains, at Asn136, 156, and 193, whereas other mammalian ATIIIs have four, additional chain being at Asn97; this is replaced by Asp in porcine ATIII. In the case of human ATIII, the chains are at Asn96, 135, 155, and 192. The heparin-binding affinities of human and porcine ATIIIs were compared using an immobilized heparin column. Porcine ATIII eluted from the column with a peak at an NaCl concentration of 924mM while human ATIII eluted at 838mM NaCl. Neuraminidase treatment of each ATIII enhanced the heparin-affinity to the same extent. These results suggest that in spite of the high degree of amino acid sequence identity between porcine and human ATIIIs (91% identical), porcine ATIII has a higher heparin-binding affinity than human, because it lacks a carbohydrate chain at Asp97.View full abstractDownload PDF (786K) -
Hiromi Sugiyama, Shunji Natori1994 Volume 116 Issue 5 Pages 1171-1175
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen the body wall of Sarcophaga larvae was pricked to activate various defense protein genes, the glutathione level in the fat body was found to decrease significantly. Previously, we showed that these defense protein genes were activated when the fat body was incubated in buffered insect saline [Sugiyama and Natori (1991) Eur. J. Biochem. 200, 495-500]. Under these conditions, the glutathione level was also found to decrease significantly. Probably, the decrease of glutathione level is crucial for activation of insect NF-κB-like factor, which has been suggested to participate in the expression of various defense protein genes.View full abstractDownload PDF (627K) -
Katsuya Nagai, Tetsuya Inazu, Hirohei Yamamura1994 Volume 116 Issue 5 Pages 1176-1181
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtein-tyrosine kinase p725syk exists abundantly in various hematopoietic cells and is activated by physiological or non-physiological agents. In this study we used vanadate, which is well known as a protein-tyrosine phosphatase inhibitor, to investigate the activity of p72syk and a downstream substrate for p72syk. Treatment with vanadate plus H2O2 caused tyrosine phosphorylation of multiple cellular proteins and platelet activation, i.e. aggregation and secretion. During aggregation induced by this stimulant, p725syk was activated and GTPase activating protein (GAP) was phosphorylated on tyrosine residue(s). The activation of p72syk was time- and dose-dependent. Also, the time course of activation of p72syk preceded that of tyrosine phosphorylation of GAP, and GAP was actually phosphorylated on tyrosine residue(s) by p72syk in vitro. These results suggest that p72syk is activated by treatment with vanadate plus H2O2, and that GAP is one of the possible substrates for p72syk in porcine platelets.View full abstractDownload PDF (4273K) -
Iwao Ohkubo, Kai Huang, Yukiko Ochiai, Masaru Takagaki, Kazutaka Kani1994 Volume 116 Issue 5 Pages 1182-1186
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDipeptidyl peptidase IV (DPP IV) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approximately 290, 000 on PAGE in the absence of sodium dodecyl sulfate (SDS) and 310, 000 on Sephacryl S-300 HR column chromatography, and to be 115, 000 and 105, 000 on SDS-PAGE in the absence and presence of β-mercaptoeth-anol. The enzyme is suggested to be composed of three identical subunits. The enzyme rapidly hydrolyzed the substrate Gly-Pro-MCA, and weakly the substrate Lys-Ala-MCA. It was strongly inhibited by diisopropylphosphofluoridate (DFP), and moderately by both phenylmethyl-sulfonyl fluoride (PMSF) and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). It was also strongly inhibited by zinc ion. The amino acid sequence of the first 18 residues of the enzyme was Asn-Lys-Gly-Thr-Asp-Asp-Ala-Ala-Ala-Asp-Ser-Arg-Arg-Thr-Tyr-Thr-Leu-Thr-. This sequence was highly homologous to the sequences in the rear of the transmembrane site of human and rat liver DPP IVs and mouse thymus DPP IV. The native DPP IV is suggested to be released into the seminal plasma after the cleavage of the hydrophobic N-terminal domain by chymotrypsin-like or pepsin-like enzymes. Other properties of DPP IV including kinetic parameters, pH stability and heat stability were characterized.View full abstractDownload PDF (1671K)
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