巻号一覧
108 巻, 2 号
選択された号の論文の31件中1～31を表示しています
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Kazuhiko Takahashi, Masami Akasaka, Yutaka Yamamoto, Chigako Kobayashi ...1990 年 108 巻 2 号 p. 145-148
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーHuman plasma glutathione peroxidase (GSHPx) has been shown to be a selenium-containing enzyme immunologically distinct from cellular GSHPx. Oligonucleotide probes, based on the partial amino acid sequence of plasma GSHPx, were synthesized and used to screen a human placenta cDNA library. Nucleotide sequence analysis of the obtained clones revealed that GSHPx consisted of a 678-base pair open reading frame coding for a 226-amino acid polypeptide with a Mr of 25, 389. About 50% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of the peptides in a lysine endopeptidase-digest of th purified enzyme. The amino acid sequence exhibited only 44% homology with that of human cellular GSHPx. Northern blot analysis revealed a single transcript of 2.2 kilobases in the poly(A)+RNA fractions of human placenta and HepG2 (a human hepatic cell line), but not that of human liver and endothelial cells.抄録全体を表示PDF形式でダウンロード (1151K) -
Kuniaki Nagayama, Toshio Yamazaki, Shigenori Kanaya, Haruki Nakamura, ...1990 年 108 巻 2 号 p. 149-152
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーA new strategy for the sequential assignment of backbone proton resonances in larger proteins involving a unique combination of four types of heteronuclear three-dimensional (3D) NMR spectroscopies is reported. This method relies on the uniform labeling of amide nitrogens with 15N and of α-carbons with 13C. Heteronuclear 1H-15N TOCSY-HMQC and NOESY-HMQC experiments can reveal connections between cross-peaks arising from the NHi-CαHi-1 and NHi-CαHi connectivities in the finger-print region in general. They also specifically reveal the sequential amide-amide connectivities among the amide cross-peaks for the α-helices. Heteronuclear 1H-13C HMQC-TOCSY and HMQC-NOESY experiments can reveal connections between cross-peaks arising from the NHi-CαHi and NHi+1-CαHi, connectivities in the finger-print region in general. The combination of the two sets of results reveals the complete unambiguous sequential connection of cross-peaks for the proton resonances in the peptide backbone. The application of the new strategy is reported for a protein, ribonuclease H, with a molecular weight of 17.6kDa.抄録全体を表示PDF形式でダウンロード (442K) -
Yozo Takasaki, Isamu Takahashi, Tsunehiro Mukai, Katsuji Hori1990 年 108 巻 2 号 p. 153-157
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーAldolase A derived from a hemolytic anemia patient with aldolase A decificency was shown to have an amino acid substitution of glycine for aspartic acid at the 128th position (Asp-128) in the enzyme [Kishi et al. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8623-8627]. We constructed an Escherichia coli expression plasmid, pHAAD128G, which carries the mutant aldolase A [aldolase A(D-G)] cDNA, and the enzyme generated in E. coli transfected with the expression plasmid was purified and characterized. Conversion of Asp to Gly at the 128th position in the enzyme rendered the enzyme thermolabile and susceptible to tryptic digestion. CD spectra analyses also revealed that the mutant enzyme had a remarkable conformation change with a decrease of regular form in the molecule. Addition of glycerol or some other polyalcohols during thermal treatment protected this altered enzyme (but not the normal enzyme) against denaturation and activity decrease. In order to determine the function of the amino acid residue at the 128th position, two artificial mutant enzymes with the substitutions of Glu for Asp [aldolase A(D-E)] and Ser for Asp [aldolase A(D-S)], respectively, at the position were constructed by site-directed mutagenesis and character-ized. These analyses demonstrated the necessity for Asp to be present at the 128th residue in order for this enzyme to be thermally stable.抄録全体を表示PDF形式でダウンロード (1839K) -
Mayumi Yoshida, Takahisa Nakai, Kenichi Fukuhara, Shin Saitoh, Wataru ...1990 年 108 巻 2 号 p. 158-165
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe three-dimensional structure of an α-amylase inhibitor, HAIM, composed of 78 amino acids, was analyzed by two-dimensional NMR techniques. Sequence-specific assignments were made for the amino acid residues from Ile-6 to Cys-72. Distance geometry analysis of the interresidue NOEs revealed that the HAIM molecule consists of two β-sheets, as is the case in a homologous α-amylase inhibitor, Tendamistat, though one of its β-strands is much shorter than that of Tendamistat. The combination of molecular modeling from Tendamistat and distance geometry analysis was confirmed to be useful for our purpose.抄録全体を表示PDF形式でダウンロード (1542K) -
Shigeru Sakajo, Nobuko Minagawa, Tadazumi Komiyama, Akio Yoshimoto1990 年 108 巻 2 号 p. 166-168
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーMitochondria exhibiting cyanide-resistant respiration were isolated from Hansenula anomala which had been incubated in the presence of antimycin A to induce cyanide-resistant respiration. The cyanide-resistant respiration in isolated mitochondria was not inhibited by antimycin A or myxothiazol, suggesting that the branching of the pathway from the normal cyanide-sensitive pathway takes place at the coenzyme Q level. Analysis of mitochondrial proteins by sodium dodecyl sulfate gel electrophoresis indicated that a 36 kDa protein was induced by antimycin A treatment of the yeast. It is suggested that this protein is a component of the cyanide-resistant respiratory pathway.抄録全体を表示PDF形式でダウンロード (1733K) -
Toshiaki Ohtsuka, Mayumi Nakamura, Masanori Hiura, Kiyomi Yoshida, Nao ...1990 年 108 巻 2 号 p. 169-174
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーTreatment of guinea pig polymorphonuclear leukocytes (PMNL) with phorbol 12-myristate 13-acetate (PMA) induced an increase in phosphorylation of 46 kDa protein(s) in parallel with activation of NADPH oxidase. In response to PMA stimulation, phosphorylated 46 kDa protein(s) increased markedly in the membrane fraction, accompanied by a decrease in the unphosphorylated form(s) in the cytosol. The results indicate that the 46 kDa protein(s) may be translocated concomitantly with its phosphorylation. On the other hand, in a cell-free activation system reconstituted from the cytosol and plasma membranes of unstimulated PMNL, arachidonic acid caused the translocation of the 46 kDa protein(s) from the cytosol to the plasma membranes concomitantly with an enhancement of NADPH oxidase activity. These results suggest that activation of NADPH oxidase is dependent on an association of 46 kDa protein(s) with the membranes both in intact PMNL and in the cell-free system.抄録全体を表示PDF形式でダウンロード (3199K) -
Shigehiro Kamitori, Akihiro Okamoto, Ken Hirotsu, Taiichi Higuchi, Sei ...1990 年 108 巻 2 号 p. 175-184
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe structure of Escherichia coli aspartate aminotransferase complex with the inhibitor 2-methylaspartate, and that of the mutant enzyme in which an arginine wassubstituted for a lysine residue thereby forming a Schiff base with the coenzyme pyridoxal 5'-phosphate, were determined at 2.5Å resolution, by the molecular replacement method using the known structure of pig cytosolic aspartate aminotransferase. The enzyme catalyzes the reversible transamination between L-aspartate and α-ketoglutarate, and forms a dimeric structure of two identical subunits. Each subunit comprises two domains, a small and a large one. Although, in general, the overall and secondary structures of E. coli enzyme are similar to those of higher animals, some differences of enzymatic action between the enzyme from E. coli and those from higher animals could be explained on the basis of the X-ray structures and molecular mechanics calculation based on them.抄録全体を表示PDF形式でダウンロード (1042K) -
Hideo Kubo, Atsushi Irie, Fuyuhiko Inagaki, Motonori Hoshi1990 年 108 巻 2 号 p. 185-192
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーNeuGcα2-6Glcβ1-1Cer (M5 ganglioside) and HS03-8NeuGcα2-6Glcβ1-1Cer (T1 ganglio-side) were purified by column chromatographies with DEAE-Sephadex A-25 and silicic acid from the eggs of the sea urchin, Anthocidaris crassispina. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, enzymatic hydrol-ysis, negative-ion fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Long-chain base compositions of both gangliosides were almost identical: all the long-chain bases were phytosphingosines, and C18-phytosphingosine accounted for more than 95% of them. Fatty acid compositions were also very similar: the main fatty acids were 22:1, 23:1, 24:1, and their 2-hydroxylated forms, and the 2-hydroxy fatty acids amounted to 65.3 and 74.3% of the fatty acids in M5 and T1 gangliosides, respectively. Proton nuclear magnetic resonance spectroscopic study revealed a down-field-shifted H8 proton signal of NeuGc residue in T1 ganglioside, in agreement with the presence of sulfate ester at the C8 position.抄録全体を表示PDF形式でダウンロード (3064K) -
Hideo Kubo, Motonori Hoshi1990 年 108 巻 2 号 p. 193-199
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーAn antibody against M5 ganglioside (NeuGcα2-6Glcβ1-1Cer), the dominant ganglioside in the eggs of the sea urchin, Anthocidaris crassispina, was purified by affinity chromatography from rabbit antiserum against crude ganglioside of the eggs. The specificity of the antibody was verified by enzyme-linked immunosorbent assay and TLC immunostaining. M5 ganglioside was also the major one in the eggs of another sea urchin, Hemicentrotus pulcherrimus, as judged from TLC analyses including immunostaining. Cryostat-sections of H. pulcherrimus eggs were examined to determine the distribution of M5 ganglioside by indirect immunofluorescence microscopy with the antibody. Before fertilization, the egg cortex was highly stained, while the other part of cytoplasm was uniformly but much more weakly stained. After fertilization, the staining rapidly decreased in the cortex and was restricted to a very thin peripheral layer and to cytoplasmic patches. The immunore-activity was also observed in the esophagus and the somatic cells of the testis, but the spermatozoa were never stained with the antibody.抄録全体を表示PDF形式でダウンロード (2888K) -
Masazumi Iwai, Masayuki Masu, Kunihiro Tsuchida, Takahide Mori, Hiroak ...1990 年 108 巻 2 号 p. 200-206
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe ISK protein is a novel, probably epithelial potassium channel which differs from conventional potassium channels in its structure, electrophysiology, and tissue distribution. In this investigation, we isolated and analyzed genomic and cDNA clones coding for the rat ISK protein to characterize the structural organization and expression pattern of the ISk protein gene. This analysis, together with primer extension and RNase protection experiments, indicated that the ISK protein mRNA is initiated from two different upstream exons and then encoded by an uninterrupted downstream exon covering the protein-coding and the 3'-untranslated regions of the mRNA. RNA blot hybridization analysis showed additional generation of several large species of mRNAs which result from inclusion of a part of the intron sequence and the 3'-flanking region of the ISK protein gene. Thus, the single ISK protein gene is involved in the production of multiple species of mRNAs through a variety of cellular mechanisms including transcription initiation at different sites, alterna-tive RNA splicing, and polyadenylation at different sites. The heterogeneity of the ISK protein mRNAs may be associated with the emergence of the functional and regulatory diversity observed for potassium ion permeation in epithelial cells.抄録全体を表示PDF形式でダウンロード (4607K) -
Kaoru Murakami, Fuminori Tokunaga, Sadaaki Iwanaga, Masataka Mori1990 年 108 巻 2 号 p. 207-214
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーOrnithine carbamoyltransferase (OTC; subunit, 36, 000 Da) [EC 2.1.3.3] is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues, then is imported posttranslationally into the mitochondrial matrix. We expressed rat pOTC in Escherichia coli, purified it in a denatured form, and showed that it could be transported into isolated mitochondria in the presence of rabbit reticulocyte lysate [Murakami et al. (1988) J. Biol. Chem. 263, 18437-18442]. In order to compare the properties of the precursor and mature form of OTC, the rat mature OTC was synthesized in E. coli and purified. The recombinant OTC represented about 5% of the total bacterial protein and was present in both the supernatant and precipitate of the disrupted bacteria. The OTC, extracted from the precipitate with 8M urea or 6M guanidine•HCl, was essentially homogeneous, as judged by SDS-PAGE. When guanidine•HCl-denatured mature OTC was diluted and incubated at 0°C for 40-60h, it was reactivated to a specific activity of 170 μmol/min/mg protein at 37°C (18% of that of the purified mature enzyme). Guanidine•HCl-denatured pOTC was activated to a specific activity of 125 μmol/min/mg protein under similar conditions. The native and reactivated OTC sedimented with an S20, w value of 6.2S, whereas the activated pOTC sedimented with an S20, w of 5.2S. The activated pOTC was more unstable than the reactivated OTC at 50°C. These observations indicate that the presequence does not prevent pOTC from folding into an enzymatically active trimeric form, although the pOTC trimer appears to be less compact than the mature trimer. Sucrose gradient centrifugation analysis showed that pOTC, but not mature OTC, formed a complex sedimenting with an S20, w, values of 6-11S in the presence of reticulocyte lysate. Formation of the complex of 35S-labeled pOTC was inhibited by unlabeled pOTC or synthetic presequence but not by mature OTC. These observations taken together with our previous findings suggest that a protein factor (s) present in the reticulocyte lysate intracts with the presequence portion of pOTC, prevents pOTC from folding, and holds it in an importcompetent form.抄録全体を表示PDF形式でダウンロード (2142K) -
Hideki Sumimoto, Emi Kusunose, Yoichi Gotoh, Masamichi Kusunose, Shige ...1990 年 108 巻 2 号 p. 215-221
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe ω-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4, ω-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) ω-hydroxylation, but not for lauric aicd ω-hydroxylation. The LTB4, ω-hydroxylation is competitively inhibited by PGA1 but not affected by lauric acid. The K1 value for PGA1 of 38 μM agrees with the Km value for PGA1 ω-hydroxylation of 40 μM. LTB4 inhibits the PGA1 ω-hydroxylation by rat liver microsomes in a competitive manner with the K1 of 43 μM, which is consistent with the Km, for the LTB4 ω-hydroxylation of 42 μM. An antiserum raised against rabbit pulmonary PG ω-hydroxylase (P-450p-s) inhibits slightly the ω-hydroxylations of LTB4, and PGA1, while it has stronger inhibitory effect on lauric acid ω-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 ω-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase.抄録全体を表示PDF形式でダウンロード (884K) -
Tarou Ogurusu, Shigeo Wakabayashi, Ken-Ichi Furukawa, Yuko Tawada-Iwat ...1990 年 108 巻 2 号 p. 222-229
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーPhosphorylation of the Ca2+-pump ATPase of cadiac sarcolemmal vesicles by exogeneously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2+-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2+-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2+-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C-kinase were also observed with a highly-purified Ca2+-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2+ pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2+-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.抄録全体を表示PDF形式でダウンロード (3330K) -
Yoshio Misumi, Miwa Sohda, Kumiko Ohkubo, Noboru Takami, Kimimitsu Oda ...1990 年 108 巻 2 号 p. 230-234
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーA cDNA clone for α1 -protease inhibitor (pcα1PI212) was isolated from a λZAP rat liver cDNA library. The 1.4 kb cDNA insert of pcα1PI212 contained an open reading frame that encodes a 411-residue polypeptide (46, 125 Da), in which a signal peptide of 24 residues was identified by comparison with the NI2-terminal sequence of the purified protein. Three potential sites for N-linked glycosylation were found in the molecule, accounting for the difference in molecular mass between the predicted form and the purified protein (56 kDa). The deduced primary structure of rat α1-protease inhibitor showed 68.5% homology to that of the human inhibitor. We then constructed the expression plasmid pSV2α1PI from pSV2-gpt and pcα1PI212, and transfected it into COS-1 cells. The transfected cells synthe-sized a molecule which was precipitated with anti-(rat α1-protease inhibitor)-IgG and had the same molecular size as that of the inhibitor produced by rat hepatocytes.抄録全体を表示PDF形式でダウンロード (1591K) -
Yasunobu Tsuji, Kenji Yamamoto, Tatsurokuro Tochikura1990 年 108 巻 2 号 p. 235-240
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーTwo types of α-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the α-(1→2), α-(1→3), and α-(1→4)-L-fucosidic linkages in various oligosaccharides and glycoproteins, but not p-nitrophenyl α-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatographies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250, 000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved α-L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl α-L-fucoside, but also 2'-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the α-(1→6)-L-fucosidic linkages to N-acetylglucosamine in 6-O-α-L-fucopyranosyl-N-acetylglucosamine, and bi- and tetra-antennary oligosaccharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.抄録全体を表示PDF形式でダウンロード (1868K) -
Yoshikazu Murawaki, Sadako Yamada, Masahiko Koda, Chisato Hirayama1990 年 108 巻 2 号 p. 241-244
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーCollagenase and collagenolytic cathepsin activities in normal and carbon tetrachlorideinduced fibrotic livers of rats were simultaneously determined at 35 and 25°C for 18 h, using the same 14C-labeled neutral soluble collagen as a substrate. Collagenolytic cathepsin had higher activity under the assay conditions at both 35 and 25°C than collagenase in normal and fibrotic livers. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the collagen was visibly degraded by collagenolytic cathepsin, but not by collagenase. These results indicate that, unlike collagenase, collagenolytic cathepsins exist as active forms in the rat liver, and can participate in the degradation of collagens, especially of soluble collagens including procollagens.抄録全体を表示PDF形式でダウンロード (1534K) -
Masao Nakamura1990 年 108 巻 2 号 p. 245-249
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe oxidation mechanism of Trolox C (a vitamin E analogue) by peroxidases was examined by stopped flow and ESR techniques. The results revealed that during the oxidation of Trolox C, peroxidase Compound II was the catalytic intermediate. The rate constants for the reaction of Compound II with Trolox C, which should be the rate-determining step, were estimated to be 2.1×104 and 7.2×103 M-1•s-1 for horseradish peroxidase and lactoperoxidase, respectively, at pH 6.0. The formation of the Trolox C radical was followed by ESR. The time course of the signal was similar to that of the optical absorbance changes at 440 nm, assigned as the peak of the Trolox C radical. The signal exhibited a hyperfine structure characteristic of phenoxyl radicals. From an estimation of the radical concentration in the steady state and the velocity of the radical formation, the dismutation constant was calculated to be 5×105 M-1•s-1. The concentration of the signal in the steady state was reduced by the addition of GSH. The spectrum changed from that of the Trolox C radical to that of the ascorbate radical when the reaction was carried out in the presence of ascorbate.抄録全体を表示PDF形式でダウンロード (503K) -
Akira Hara, Hiroyuki Taniguchi, Toshihiro Nakayama, Hideo Sawada1990 年 108 巻 2 号 p. 250-254
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーTwo acidic and three basic forms of monomeric dihydrodiol dehydrogenase with molecular weights in the range of 36, 000-39, 000 were purified from human liver. One acidic enzyme (pI 5.2), which was specific for NADP+ and dihydrodiols of benzene and naphthalene, was immunologically identified as aldehyde reductase. The other four enzymes oxidized alicyclic alcohols as well as the dihydrodiols using both NADP+ and NAD+ as cofactors, but showed differences in specificity for hydroxysteroids and inhibitor sensitivity. Two of the basic enzymes (pI 9.7 and 9.1) exhibited a 20α-hydroxysteroid dehydrogenase activity and sensitivity to 1, 10-phenanthroline, whereas the third basic enzyme (pI 7.6) oxidized some 3α-hydroxysteroids at low rates and was inhibited by cyclopentane-1, 1-diacetic acid. Another acidic enzyme, which accounted for the largest amount of enzyme activity in the tissue and appeared in two heterogenous forms with pI values of 5.9 and 5.4, showed a high 3a-hydroxysteroid dehydrogenase activity and was the most sensitive to inhibition by medroxyprogesterone acetate. The Km values of the enzymes, except the pI 5.2 enzyme, for hydroxysteroids (10-6 to 10-7M) were lower than those for xenobiotic alcohols.抄録全体を表示PDF形式でダウンロード (1545K) -
Yasuko Abe, Toshiharu Ito, Taro Okazaki1990 年 108 巻 2 号 p. 255-260
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーTwo protein components having a NADPH-dependent methemoglobin reductase activity were purified to electrophoretic homogeneity from the erythrocytes of the bullfrog, Rana catesbeiana. Their molecular properties were investigated. The components were separat-ed by isoelectric focusing, having discrete bands of pI 5.0 and 7.5, respectively. The pI 5.0 component, designated F-5.0, was faint yellow, with a broad absorption in the range of 400-450nm, while the pI 7.5 component, designated F-7.5, was colorless and did not absorb in that range. The molecular weight was estimated to be 22, 000 for both components by gel filtration and SDS-PAGE. When F-5.0 was subjected to isoelectric focusing repeatedly, the protein part of that component gradually moved to and refocused at pH 7.5, leaving a yellow color at acidic pH. Both F-5.0 and F-7.5 were highly specific for NADPH and had the same kinetic properties in catalyzing the reduction of MB, DCPIP, FMN, or FAD, and that of methemoglobin or cytochrome c in the presence of a certain dye. They were also indistin-guishable from one another in their amino acid compositions and were completely identical in the N-terminal sequence of 24 amino acid residues. These findings strongly suggest that the two components can be attributed to the same enzyme molecule, carrying an identical protein moiety but interacting differently with some unidentified biological pigments, and that they are equivalent in their molecular and kinetic properties to the NADPH-dependent enzyme(s) occurring in human erythrocytes.抄録全体を表示PDF形式でダウンロード (1502K) -
Tatsushi Muta, Tamiji Fujimoto, Hiroshi Nakajima, Sadaaki Iwanaga1990 年 108 巻 2 号 p. 261-266
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーTachyplesins and their analogs are antimicrobial peptides composed of 17 or 18 amino acid residues present abundantly in acid extracts of hemocyte debris of horseshoe crabs. We purified here tachyplesin isopeptides from hemocytes of two species of Southeast Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus gigas, and determined their amino acid sequences. The major tachyplesin isolated from both species was identified, respectively, as tachyplesin I, which had previously been found in hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus). The yield from both species was very high (more than 70 mg per 100 g wet weight of hemocytes), i.e., comparable with that from T. tridentatus. In addition to tachyplesin I, a new tachyplesin isopeptide, named tachyplesin III, was also isolated from T. gigas hemocytes, in which an arginine replaced the 15th lysine of tachyplesin I. The carboxyl-terminal residue of the isolated tachyplesins I and III was confirmed, respectively, to be an arginine α-amide by chemical analysis. Furthermore, a tachyplesin peptide derivative with a carboxyl-terminal extension of glycine-lysine was newly found in the hemocytes of C. rotundicauda. It appeared to be an intermediate derived from a tachyplesin precursor during processing to the mature form.抄録全体を表示PDF形式でダウンロード (646K) -
Kazuo Miyoshi, Yoshiko Egi, Toshihiro Shioda, Takashi Kawasaki1990 年 108 巻 2 号 p. 267-270
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーWe showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphos-phate (TDP) and ADP in addition to ATP formation from ADP [Shikata, H. et al. (1989) Biochem. Int. 18, 933-942]. To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching. Thiamin phosphate metabolism in chicken skeletal muscle was also studied. i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle. Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle. ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained. Both AK1 activity and TTP- synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum. iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant. In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching. These results suggest in vivo synthesis of TTP by AK1. iv) The TTP/TDP ratio on day 2 after hatching was nearly the same (0.19-0.34) in both types of muscle, and then increased to 3.7 in the white muscle on day 16 after hatching, but remained almost constant in the red muscle. These results suggest that in the chicken, TTP metabolism in the white muscle is initially the same as that in the red muscle just after hatching, and then develops into the white muscle type in the following 2 weeks.抄録全体を表示PDF形式でダウンロード (475K) -
Yohtalou Tashima, Miyuki Terui, Hideaki Itoh, Hideo Mizunuma, Ryoji Ko ...1990 年 108 巻 2 号 p. 271-277
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーWe purified glucocorticoid receptors quickly but very partially using DEAE-resin. [3H]-Triamcinolone acetonide-labeled and non-activated receptors in the quickly purified fraction were found to be separated into two fractions (P-2 and P-3) by hydroxyapatite column chromatography. The P-2 receptor was the main component, and the ratio of P-2/ P-3 was around 2. The molecular weights of the two receptors were calculated to be the same, 242, 000:RS=6.2 nm and s20, W=9.0. Treatment of the receptor with catalytic subunits of phosphoprotein phosphatase 2A1 reduced the P-2/P-3 ratio from 2 to 0.5, while treatment with catalytic subunits of cAMP-dependent'protein kinase and ATP incresed it to 2.5. The isolated P-3 receptor could be converted into the P-2 type by the kinase treatment. Tungstate, a phosphatase inhibitor, stabilized the P-2 receptor, and the P-2/P-3 ratio was larger than 3 when the DEAE-fraction was prepared in the presence of tungstate. However, the tungstate effect was not very strong, and the P-2 type tended to change into the P-3. [3H]-Triamcinolone acetonide-labeled and non-activated receptors were purified very highly by using an affinity gel; the precedure required more than 10 h. Only the P-3 form was observed in the preparation of highly purified receptors. Hormone-free receptors were affected by neither the phosphatase nor the kinase. The results indicate that the hormone binding makes the receptor sensitive to phosphatase. The reversibly dephosphorylated receptor is more stable than the non-dephosphorylated one, and can be activated.抄録全体を表示PDF形式でダウンロード (835K) -
Yoshitaka Tanaka, Ryoko Harada, Masaru Himeno, Keitaro Kato1990 年 108 巻 2 号 p. 278-286
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-β-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloro-quine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1 + 2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultane-ously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region. At 3 h after the injection, acid phos-phatases of 64 and 55 kDa were detected in lysosomal content fractions. Moreover, at 12 h after the injection, an acid phosphatase of 48 kDa, as well as the 55 kDa form, was present in the lysosomal content fraction, thereby indicating that after transport to the lysosomes, the membrane-bound acid phosphatase of 67 kDa was released into the lysosomal matrix, as the 64 kDa form, and was subsequently processed to the 55 kDa form and then converted into the 48 kDa form. The processing of the 67 kDa membrane-bound form to the 48 kDa soluble form involved removal of the protein portion and of the carbohydrate moieties. These results are interpreted to mean that acid phosphatase is targeted to the lysosomes in a manner independent of the mannose-6-phosphate system.抄録全体を表示PDF形式でダウンロード (5840K) -
Yoshitaka Tanaka, Masaru Himeno, Keitaro Kato1990 年 108 巻 2 号 p. 287-291
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーWe examined the mechanism of release of acid phosphatase (APase) from lysosomal membranes into the lysosomal matrix. When rat liver lysosomal membranes were incubat-ed at various pH values with APase-free tritosomal contents prepared by the treatment of tritosomal contents with anti-APase IgG Sepharose, 86% of the APase activity in the lysosomal membranes became soluble at pH 5.0. Immunoblots revealed that the membrane-bound APase (67 kDa) was released in a 64 kDa form, and the 67 and 64 kDa forms were converted to 45 and 41 kDa forms by Endo F treatment, respectively, thereby indicating that the release of APase from the lysosomal membranes was accompanied by a limited proteolysis involving loss of a 4 kDa fragment. The release of APase was strongly inhibited by pepstatin A, a potent inhibitor of aspartyl protease, but other inhibitors such as leu-peptin, antipain, Ep-475 and 1, 10-phenanthroline showed no effect. The release of APase did not occur when the lysosomal membranes were incubated with the tritosomal contents free of APase and cathepsin D, prepared by treatment of the APase-free tritosomal contents with anti-cathepsin D IgG Sepharose. The purified lysosomal cathepsin D released 71% of the APase activity from the lysosomal membranes and the released APase had a molecular mass of 65 kDa, that is, larger than the enzyme released by using the APase-free tritosomal contents. Endo F converted the 65 kDa form to the 43 kDa form. When the lysosomal membranes were incubated at pH 5.0 with the APase-free tritosomal contents in the presence of leupeptin, antipain, and Ep-475, the 65 kDa form instead of the 64 kDa form was observed as a soluble enzyme. Taken together, these results show that APase is released from lysosomal membranes into the lysosomal matrix by cathepsin D in the 65 kDa form, with release of a 2 kDa peptide, following which the released enzyme is further processed to the 64 kDa form, probably by lysosomal cysteine protease.抄録全体を表示PDF形式でダウンロード (4421K) -
Hirotada Fujii, Katsuko Kakinuma1990 年 108 巻 2 号 p. 292-296
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーSuperoxide (O2-)-generating membranes of pig blood neutrophils were studied by the ESR spin-label method. Neutrophils were spin-labeled with doxylstearic acids, consisting of nitroxide free radicals bonded to the 5, 7, 12, or 16 position of stearic acid (5-, 7-, 12-, or 16-DS), to detect the reduction of their nitroxide radicals at different positions in the membrane. The spin-labeled cells were then stimulated with phorbol myristate acetate (PMA). Stimulation of the labeled cells resulted in a marked decrease in the spin concentra-tion of 5-DS due to the reduction by O2-, but not in those of the other three DS labels. This reduction of 5-DS was completely inhibited by copper salicylate (CS), a hydrophobic and permeable O2--scavenger, but not by superoxide dismutase (SOD). CS was not inhibitory on the respiratory burst, i.e., O2--generating activity of neutrophils. On the contrary, if the spin-labels were present in the extracellular medium, SOD inhibited the reduction of all four DS labels due to O2-released from PMA-stimulated cells. These results suggest that the O2--releasing site is not located at the outer surface of the plasma membrane but in an inner hydrophobic environment a short distance (around 4-5 A) from its outer surface.抄録全体を表示PDF形式でダウンロード (599K) -
Tatsuhiko Furukawa, Masayuki Ozawa, Ruo-Pan Huang, Takashi Muramatsu1990 年 108 巻 2 号 p. 297-302
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーA cDNA clone isolated from a λgt11 expression library of teratocarcinoma OTT6050 specifies for a glycoprotein with a molecular weight of about 44, 000. The new glycoprotein was termed heparin binding protien - 44 (HBP-44), since it was absorbed to a heparin-agarose column and was eluted from it by a buffer containing 1.5 M NaCl. HBP-44 mRNA was intensely expressed in PYS-2 parietal endoderm cells and in the kidney, and the RNA level increased about 10-fold during differentiation of F9 embryonal carcinoma cells to parietal endoderm cells. From the cDNA sequence, HBP-44 was concluded to be rich in charged amino acids, and large segments of the protein appeared to form α-helixes. The protein was considered to be anchored to the membrane by a cluster of hydrophobic amino acids present in the N-terminal region. Indeed, the N-terminal sequence of HBP-44 was homologous to asialoglycoprotein receptor, which is anchored to the membrane by the N-terminal region. Furthermore, a portion of the N-terminal region of HBP-44 was homologous to the leucine zipper domain. Except for the N-terminal region, HBP-44 had over-all homology with structural proteins such as myosin heavy chain. We propose that IIBP-44 is extruded from plasma membranes and interacts with heparin and related molecules and that it is involved in the interactions of plasma membranes with basement membranes.抄録全体を表示PDF形式でダウンロード (2820K) -
Hideaki Watanabe, Akiko Naitoh, Yuka Suyama, Norio Inokuchi, Hiroko Sh ...1990 年 108 巻 2 号 p. 303-310
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase M) from Aspergillus saitoi was determined. The sequence was determined by analysis of the peptides generated by digestion of heat-denatured RNase M with lysylen-dopeptidase, and the peptides generated from RCM RNase M by digestion with staphylococ-cal V8 protease or chemical cleavage with BrCN. It consisted of 238 amino acid residues and carbohydrate moiety attached to the 74th asparagine residue. The molecular weight of the protein moiety deduced from the sequence was 26, 596. The locations of 10 half cystine residues are almost superimposable on those of RNase Rh from Rhizopus niveus and RNase T2 from Aspergillus oryzae which have similar base specificity. The homology between RNase M and RNase Rh and RNase T2 amounted to 97 and 160 amino acid residues, respectively. The amino acid sequences conserved in the three RNases are concentrated around the three histidine residues, which are supposed to form part of the acitve sites of these RNases.抄録全体を表示PDF形式でダウンロード (820K) -
Takako Morimoto, Chikara Oho, Masahiro Ueda, Satoshi Ogihara, Haruhiko ...1990 年 108 巻 2 号 p. 311-320
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーMicromolar levels of free calcium ions added to the extracellular medium elicit secretion of serotonin from electropermeabilized bovine platelets in the presence of millimolar levels of Mg-ATP. Such Ca2+-dependent secretion of serotonin was almost completely impaired when the permeabilized platelets were preincubated for 1 min at 35°C in 100μM Ca2+ without Mg-ATP. The half-maximal effect was observed with about 45μM Ca2+ in the preincubation medium. Inhibitors of serine-thiol protease, such as leupeptin and antipain, suppressed the impairment of the secretion of serotonin by the preincubation with Ca2+. Electron microscopic observation revealed that disorganization of the cytoskeletal struc-tures, in particular of the membrane undercoat and the network of microfilaments, accompanied the impairment of secretion of serotonin. Microfilaments were also found to be dissociated from dense granules that contained serotonin. These morphological changes were also suppressed when antipain was included in the Ca2+-preincubation medium. Coincident with these morphological changes, the following biochemical changes were observed in 100μM Ca2+ but not in the presence of Ca2+ and antipain. The amount of Triton-insoluble cytoskeleton and the acto-myosin content of the dense-granule fraction were markedly decreased. The decrease in Triton-insoluble cytoskeletons was quantita-tively correlated with the degree of impairment of secretion of serotonin. Immunoblot analysis of EGTA extracts of the cells showed that the 240-kDa spectrin in platelets was degraded to a 235-kDa fragment, and a 260-kDa actin-binding protein (ABP) in platelets was partially degraded to 190- and 110-kDa components. A novel 88-kDa protein in the dense-granule fraction, which was shown to be an actin-binding protein by affinity chromatography on a DNase 1 column, was also found to be cleaved upon preincubation with Ca2+ in the absence of antipain. These results indicate that the Ca2+-dependent secretion of serotonin is impaired, and that the cytoskeletal structures are at the same time seriously damaged through the activation of an endogenous protease activated by Ca2+. Three actin-binding proteins, namely, spectrin, ABP, and an 88-kDa protein, were identified as substrates for the protease. They might play some positive roles in the secretion of serotonin from platelets.抄録全体を表示PDF形式でダウンロード (5866K) -
Takuya Katsube, Yasuaki Kazuta, Hiroyuki Mori, Kenichi Nakano, Katsuyu ...1990 年 108 巻 2 号 p. 321-326
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーWe have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1, 758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1, 431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemi-cally [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51, 783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homo-logous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coll.抄録全体を表示PDF形式でダウンロード (733K) -
Kiyofumi Maruyama1990 年 108 巻 2 号 p. 327-333
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリー4-Hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] has been purified to homogeneity (about 770-fold purification, yield 11.4%) from Pseudomonas ochraceae grown on phthalate. The enzyme has a molecular weight of 160, 000 (gel filtration on Bio-Gel A-1.5m), a subunit molecular weight of 26, 000 (SDS-PAGE) and an isoelectric point of 5.0 (isoelectric focusing). The enzyme requires divalent metal ions such as Mg2+, Mn2+, Co2+, Zn2+, and Cd2+ for activity. The enzyme actively cleaves 4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, to give pyruvate and oxaloacetate, but shows much lower affinity for 4- hydroxy-4-methy1-2-oxoglutarate. 4-Hydroxy-2-oxoglutarate is cleaved at a low rate to pyruvate and glyoxylate. The l-isomers of the substrates are preferentially cleaved rather than the d-isomers as determined polarimetrically. The enzyme reactions are reversible: the equilibrium constants (pH 8.0, 25°C) for the HMG and HG cleavage reactions are about 0.07 and 0.03 M, respectively, whereas no equilibrium is observed with CHA due to oxaloacetate β-decarboxylase activity associated with the enzyme. The enzyme activity is hardly affected by thiols and thiol reagents. The non-enzymatic cleavage reaction caused by various metal ions has also been studied to examine the mechanistic similarity to the enzymatic reaction.抄録全体を表示PDF形式でダウンロード (924K) -
Kiyofumi Maruyama1990 年 108 巻 2 号 p. 334-340
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーPseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4- methy1-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17], one of the metal ion-requiring aldolases, is markedly activated by P1. The activation is reversible and can be observed in every step of enzyme purification. The extent of activation is almost independent of the metal ion used, but varies with each substrate. The cleavage of l-4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, is most strongly activated: P1 gives a hyperbolic activation curve with an activation constant of 0.36 mM and a maximum activation of about 65-fold. Arsenate, phosphorous acid, bicarbonate, acetyl phosphate, thiamine diphosphate, ADP, PP1, and ATP are also effective to various extents. These anions appear to be effective in the free form but not in the metal ion-complex. Many organic and inorganic anions are ineffective. P1 causes parallel increases in Vmax and in Km for substrate or metal ion with a concomitant shift of the optimum pH toward the alkaline side, and the enhancement of activity is closely correlated with the shift of optimum pH. P1 induces no gross change of molecular form of the enzyme protein as evaluated from gel filtration, PAGE, UV, fluorescence, and CD spectral data. Based on these findings, the mechanism and the physiological meaning of the observed activation are discussed.抄録全体を表示PDF形式でダウンロード (810K)
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