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126 巻, 5 号
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Takaharu Yamamoto, Shinichiro Taya, Kozo Kaibuchi1999 年 126 巻 5 号 p. 799-803
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーRas is a signal-transducing, guanine nucleotide-binding protein for various membrane receptors including tyrosine kinase receptors. Ras participates in the regulation of cell proliferation, differentiation, and morphology. Activated ras oncogenes have been identified in various forms of human cancer including epithelial carcinomas of the lung, colon, and pancreas. The cells of these cancers, as well as those that have been experimentally transformed by the activated ras gene, exhibit abnormal growth, morphological changes and alterations of cell adhesions. Although the main effector protein has been thought to be Raf serine/threonine kinase, research has revealed that the Ras-induced signaling pathway is mediated by multiple effector proteins and has the crosstalk with various factors containing other small GTPases. In this review, we summarize the involvement of each effector protein for Ras and the crosstalk with other small GTPases in Ras-induced transformation.抄録全体を表示PDF形式でダウンロード (461K) -
Ikuko Takahashi, Youko Nakamura, Yuri Hamada, Kiyoshi Nakazawa1999 年 126 巻 5 号 p. 804-814
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーAntibodies to core proteins of chicken corneal keratan sulfate proteoglycan and chondroitin sulfate proteoglycan were prepared and purified by use of an affinity column. Using these antibodies and monoclonal antibody 5-D-4 to keratan sulfate (commercial), the localization of proteoglycans in developing corneas (Days 5 to 17 of embryonic age and 2 days after hatching) was determined immunohistochemically. Keratan sulfate proteoglycan antigen was not detected in cornea on Day 5, but it was detected uniformly over the whole stroma on Day 6, ca. 12 h after invasion of the primary stroma by mesenchymal cells. The absence of the antigen in cornea of Day 5 was confirmed by Western blotting of the corneal extract. Immunohistochemistry with 5-D-4 antibody revealed that the keratan sulfate chain was undersulfated in corneas of Days 6 to 7, because the staining was much weaker than that in cornea of Day 8. In addition, keratan sulfate proteoglycan antigen was detected uniformly over the whole stroma on Days 7 to 17 and 2 days after hatching, but not in the epithelial layer on Day 13 and after: because the epithelial layer was clearly not observed on photomicrographs until Day 13, it is not known whether keratan sulfate proteoglycan was synthesized by the epithelium during Days 6 to 12. In contrast, chondroitin sulfate proteoglycan antigen was detected in cornea on Day 5 and also, like keratan sulfate proteoglycan, uniformly over the whole stroma on Day 6 through 2 days after hatching. Furthermore, the chondroitin sulfate proteoglycan was not detected in the epithelial layer on Day 13 and after. These results show that keratan sulfate proteoglycan is synthesized by the stromal cells which invade the primary stroma between Day 5.5 and 6, while chondroitin sulfate proteoglycan is synthesized by epithelial and/or endothelial cells before the invasion, and also by the stromal cells after the invasion.抄録全体を表示PDF形式でダウンロード (3660K) -
Seiji Tsuboi1999 年 126 巻 5 号 p. 815-820
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーHepatocyte growth factor (HGF) was found to cause a dose- and time-dependent increase in intracellular glutathione (GSH) level (2.0-fold in 24 h with 5 ng/ml) in rat hepatocytes. The activity of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme of GSH biosynthesis, was also increased by HGF (1.7-fold in 24 h with 5 ng/ml). Northern blot analysis revealed the induction of γ-GCS mRNA. These increases by HGF were synergically stimulated by the addition of insulin. In contrast, epidermal growth factor had no effect on GSH level and γ-GCS activity in hepatocytes. These results suggested that the induction of GSH synthesis by HGF is associated with the transcriptional activation of the γ-GCS gene and the subsequent elevation of γ-GCS activity.抄録全体を表示PDF形式でダウンロード (1132K) -
Takeshi Shimomura, Kimitoshi Denda, Toshiya Kawaguchi, Kouji Matsumoto ...1999 年 126 巻 5 号 p. 821-828
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーHepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor, which was identified as a potent inhibitor of hepatocyte growth factor (HGF) activator from the conditioned medium of a human carcinoma cell line. HGF activator is a blood coagulation factor XII-like serine protease that is responsible for proteolytic activation of the inactive single chain precursor of HGF in injured tissues. The predicted sequence of the primary translation product of HAI-1, which has a hydrophobic sequence in its COOH-terminal region, suggested that HAI-1 is first produced in a membrane-associated form. In this study, we identified a transmembrane form of HAI-1 integrated in the plasma membrane of cultured cells using a monoclonal antibody against HAI-1. We also identified several soluble forms of HAI-1 in the conditioned medium of the cells, indicating that multiple sites are present in the transmembrane form of HAI-1 at which proteolytic cleavage releases the extracellular domain. At least two proteases, one of which is a metalloprotease, appear to be responsible for the release. Further, the soluble forms of HAI-1 have different inhibitory activity against HGF activator. These findings suggest that proteolytic processing plays important roles in regulation of the inhibitory activity of HAI-1.抄録全体を表示PDF形式でダウンロード (2831K) -
Hiroyuki Hori, Toshio Iwasaki, Yoko Hayashi, Yoko Kurahashi, Tomohiro ...1999 年 126 巻 5 号 p. 829-837
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーPhosphatidylinositol 4, 5-bisphosphate (PIP 2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS; EC 1. 14. 13. 39). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent Km for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP 2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca2+-calmodulin was observed in the presence of PIP 2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca2+-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP 2 and PA. These results strongly suggest that PIP 2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca2+-calmodulin.抄録全体を表示PDF形式でダウンロード (2859K) -
Takuya Takahashi, Sachiko Hiramoto, Shin-ichi Wato, Tomoyuki Nishimoto ...1999 年 126 巻 5 号 p. 838-844
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーKidney bean (Phaseolus vulgaris) α-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (αβ)_??_ complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) α-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate α-amylase revealed the involvement of two tryptophan residues in α-amylase inhibition, and they were identified as Trp 188 of the β-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr 186 of the β-subunit as an essential residue.抄録全体を表示PDF形式でダウンロード (1539K) -
Fumiko Aoki, Kazuhiko Nakabayashi, Yusuke Wataya, Shiro Kankawa, Sumik ...1999 年 126 巻 5 号 p. 845-851
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーUpon shift-up in temperature, mouse tsFS 20 mutant cells with thermolabile ubiquitinactivating enzyme E 1 immediately stopped DNA replication and showed cell cycle arrest in S-phase. In contrast, when the cells were permeabilized with lysolecithin after culture at the nonpermissive temperature, they exhibited a normal level of repliative DNA synthesis in vitro. In agreement with this, intracellular pools of deoxyribonucleoside triphosphates were significantly reduced in the cells cultured at the nonpermissive temperature. Even under the permissive conditions, tsFS 20 cells were more sensitive to hydroxyurea and alkylating agents, and induced less mutation than the wild-type cells. These results suggest that the ubiquitin system affects DNA replication and repair.抄録全体を表示PDF形式でダウンロード (591K) -
Seiji Ohashi, Kazuma Iwai, Tomohiro Mega, Sumihiro Hase1999 年 126 巻 5 号 p. 852-858
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe amounts and isomeric structures of free oligosaccharides derived from N-linked sugar chains present in the cytosol fraction of perfused mouse liver were analyzed by tagging the reducing end with 2-aminopyridine followed by 2-dimensional HPLC mapping with standard sugar chains. Sixteen pyridylaminated (PA-) oligomannosides terminating with a PA-GlcNAc residue (GN 1-type), three glucose-containing oligomannosides, and four oligomannosides terminating with a PA-di-N-acetylchitobiose (GN 2-type) were detected. The total contents of the GN 1- and GN 2-type oligomannosides were 3.4 and 0.5 nmol, respectively, per gram of wet tissue. Maltooligosaccharides (dimer to pentamer) were also detected, the total content of which was 13 nmol per gram of wet tissue. Besides these oligosaccharides, a PA-disialobiantennary sugar chain-the sole complex-type sugar chain -was also detected. All the oligomannosides identified had partial structures of Glc3Man9GlNAc2-p-p-dolichol, revealing that they were metabolic degradation products. Manα1-2 Manα1-2 Manα1-3 (Manα1-6) Manβ1-4 GlcNAc (M 5 B') was the major oligomannoside, suggesting that cytosolic endo-β-N-acetylglucosaminidase and neutral α-mannosidase participate in the degradation, because these enzymes have suitable substrate specificities for the production of M 5 B'. Degradation by these enzymes seems to be the main pathway by which oligomannosides are degraded in mouse cytosol; however, small amounts of Manα1-6 (Manα1-3) Manα1-6 (Manα1-3) Manβ1-4 (GlcNAc)1-2 and related oligomannosides together with parts of their structures were also detected, suggesting that there is another minor route by which cytosolic free oligomannosides are produced.抄録全体を表示PDF形式でダウンロード (575K) -
Hiroaki Sasakawa, Atsuo Tamura, Shinichi Fujimaki, Seiichi Taguchi, Ka ...1999 年 126 巻 5 号 p. 859-865
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーBased on the nuclear magnetic resonance assignments of a dimeric protein, Streptomyces subtilisin inhibitor (SSI), microscopic details of secondary structures in solution have been elucidated. The chemical shift index of C_??_ signals, together with information on the hydrogen exchange rates of the backbone amide protons, were used to identify secondary structures. The locations of these secondary structures were found to be different in some critical points from those determined earlier by X-ray crystallography of the crystal. Notably, the β3 strand is completely missing and the α2 helix is extended toward the C-terminus. Furthermore, hydrogen exchange experiments of individual peptide NH protons under strongly folding conditions revealed mechanisms of global and local structural fluctuation within the dimeric structure. It has been suggested that the global fluctuation of the monomeric unit occurs without affecting the accompanying monomer, in contrast to the equilibrium thermal unfolding, which is cooperative. Higher protection against hydrogen exchange for residues in part of the β4 strand implies that this region might serve as a folding core.抄録全体を表示PDF形式でダウンロード (2928K) -
Akihito Yasuoka, Kiyoshi Endo, Misaki Asano-Miyoshi, Keiko Abe, Yasufu ...1999 年 126 巻 5 号 p. 866-873
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーWe cloned and characterized two subfamilies of olfatory receptor (OR) genes from medaka fish (Oryzias latipes). Southern blot analysis showed that each of the two subfamilies, designated as subfamilies Y and E, consists of about five members, as usually observed for other vertebrate ORs. Analyses of the genomic clones encoding these members revealed that two members of subfamily Y and four members of subfamily E are tandemly reiterated in 15 and 22 kbp regions of the medaka genome, respectively. The members of each subfamily show very similar amino acid sequences, with similarities greater than 70%. However, the similarities to the sequences of other vertebrate ORs are lower. Members of subfamily Y show amino acid sequence similarities of ca. 30% to other fish ORs, including subfamily E members, as well as to mammalian ORs. On the other hand, members of subfamily E show sequence similarities of ca. 50% to other fish ORs and ca. 30% to mammalian ORS. Phylogenic analyses of various fish ORs, including medaka, catfish, and zebrafish ORs, indicate that the primary structures of fish ORs are diverse compared with those of mammalian ORs, which consist of much larger numbers of members. The expression patterns of subfamilies Y and E genes in the olfactory epithelium of adult medaka fish were examined by in situ hybridization, showing that the frequency of positive signals is different between the two subfamilies: about 2% of the olfactory neurons are positive to probes for members of subfamily Y, while less than 1% are positive to probes for members of subfamily E. These results indicate that each subfamily is under different transcriptional control.抄録全体を表示PDF形式でダウンロード (2190K) -
Kaori Moriwaki, Tadashi Ogishima, Akio Ito1999 年 126 巻 5 号 p. 874-878
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーWe recently demonstrated, using synthetic peptides modeled on the extension peptide of malate dehydrogenase, that amino acid residues present at the proximal and distal positions relative to the cleavage site are critical determinants for the recognition of substrates by mitochondrial processing peptidase [Niidome et al. (1994) J. Biol. Chem. 269, 24719-24722]. While the proximal arginine is unexceptionally located at the -2 position, the position of the distal residue varies among mitochondrial precursor proteins. Between the proximal and distal residues, praline and/or glycine are present in most mitochondrial precursor proteins, and they are considered to play a role in the specific recognition of a substrate by the peptidase. To elucidate the role of the intervening portion, we introduced a non-natural amino acid [2-(2-aminoethoxy) acetic acid] between the distal and proximal residues. We also analyzed the functional elements in the proximal arginine by replacing the residue with various arginine or lysine analogs. The results of kinetic studies indicated that the intervening portion should be flexible for efficient processing, and that the guanidino group of the proximal arginine is recognized by the peptidase through hydrogen and ionic bonds.抄録全体を表示PDF形式でダウンロード (494K) -
Ryousuke Himukai, Takashi Kuzuhara, Masami Horikoshi1999 年 126 巻 5 号 p. 879-888
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe Schizosaccharomyces pombe gene, fkp 39+, encoding a homolog of FKBP (FK 506 binding protein)-type peptidyl prolyl cis-trans isomerase (PPIase), was isolated and the primary structure was determined. This gene product (SpFkbp 39 p) showed PPIase enzymatic activity in a chymotrypsin-dependent enzyme assay involving recombinant SpFkbp 39 p. Comparison of the primary structures of the catalytic domains of FKBPs, including SpFkbp 39 p, revealed that FKBPs could be classified into four groups. This categorization corresponding to the known subcellular localization of the FKBPs, makes the prediction of the subcellular localization of FKBPs based on their primary structures feasible. SpFkbp 39 p was considered to be a member of the nuclear-type FKBP group from this relationship beween primary structure and subcellular localization. An immunofiuorescence assay against HA-epitope-tagged SpFkbp 39 p revealed that SpFkbp 39 p is localized to the nucleus, as predicted. Residues conserved in a “group-specific” manner in the catalytic domain were mapped to their corresponding three-dimensional positions; these “group-specific” residues were located in close proximity in distinct regions mostly on the protein surface, which implies the presence of “group-specific” regulatory functional regions. We also found that nuclear-type FKBPs, including SpFkbp 39 p, have two highly conserved domains other than catalytic ones, with further basic and acidic charged regions, especially in the case of nuclear-type FKBPs. This is the first report indicating that there is a rule for the relationship between the subcellular localization and structure of the catalytic domain of a FKBP.抄録全体を表示PDF形式でダウンロード (3740K) -
Taketomo Minoshima, Shigetada Nakanishi1999 年 126 巻 5 号 p. 889-896
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーMetabotropic glutamate receptors (mGluRs) belong to the class of G protein-coupled receptors and consist of eight different subtypes. We have characterized the structural organization of the mouse mGluR 3 gene by genomic cloning in combination with rapid amplification of 5'- and 3'-cDNA ends and examined regulatory expression of mGluR 3 mRNA in cultured cortical astrocytes. The mGluR 3 gene consists of six exons and spans over 95 kb. Exon 1 and its preceding putative promoter are located distantly from the following protein-coding region. In the mGluR family, mGluR 3 and mGluR 5 are both expressed in neuronal and glial cells and are upregulated during the early postnatal period. They are, however, coupled to two distinct signaling cascades and have been shown to exert opposite influences on some functions of cultured astrocytes. In cultured astrocytes, mGluR 3 and mGluR 5 mRNA levels were significantly increased by exposure to epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or transforming growth factorα; and EGF was more efficacious than bFGF in producing this increase. Hence, mGluR 3 and mGluR 5 mRNAs are concertedly upregulated in cultured astrocytes by specific growth factors. This finding suggests that the two mGluR subtypes may play an important role in maintaining the proper balance of astrocyte functions via two distinct signal transduction mechanisms.抄録全体を表示PDF形式でダウンロード (1531K) -
Hiroshi Miyoshi, Takeshi Youtani, Hiroko Ide, Hiroyuki Hori, Ken Okamo ...1999 年 126 巻 5 号 p. 897-904
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーA translation initiation factor, eIF 4 E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF 4 E and 4 E-BP 2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF 4 E with an mRNA cap-analogue enhanced the binding activity of eIF 4 E with 4 E-BP 2. Furthermore, the SPR analysis showed that the eIF 4 E cap-analogue interaction was very weak regardless of complex formation of 4 E-BP 2 with eIF 4 E; the dissociation constant of eIF 4 E for the cap-analogue was estimated to be 10-2-10-4 M. These results suggest that the participation of another initiation factor is required for eIF 4 E to recognize the cap structure in vivo. The results reported in this paper support “the performed complex model” of Lee et al., in which eIF 4 E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF 4 F.抄録全体を表示PDF形式でダウンロード (4381K) -
Hiroshi Oneda, Kuniyo Inouye1999 年 126 巻 5 号 p. 905-911
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe recombinant prepro-form of human matrix metalloproteinase 7 (matrilysin or MMP-7) was overexpressed in Escherichia coli as insoluble inclusion bodies. The recombinant protein was refolded by 100-fold dilution after solubilization with 6M guanidine HCl. The refolding was monitored by the recovery of matrilysin activity. The addition of either 1.0M arginine or 0.1% Brij-35 promoted remarkably the refolding. The refolding was dependent on pH and temperature, with lower temperature (<10°C) and pH 6-8 preferable. Glutathione had no effect on refolding, and it was excluded from the refolding conditions. Starting with inclusion bodies (2.0g, wet) containing 360mg protein, 29.5mg of promatrilysin (30 kDa) was obtained after refolding with 1.0% Brij-35 at pH 7.5 and 4°C for 12 h. Pro-matrilysin (24.0mg) was purified to homogeneity by cation-exchange HPLC with a 15-fold increase in purity and an activity yield of 81.3%. Pro-matrilysin was converted entirely to matrilysin (19.0 kDa; 15.2mg) by activation with a mercuric reagent. The activity (kcat/Km) of matrilysin was 1.7×105M-1•s-1.抄録全体を表示PDF形式でダウンロード (3044K) -
Ryoko Iijima, Shinji Yamaguchi, Ko-ichi Homma, Shunji Natorit1999 年 126 巻 5 号 p. 912-916
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーWe examined the effects of various protease inhibitors on Xenopus laevis embryogenesis. Aprotinin, a serine protease inhibitor, was found to inhibit embryogenesis markedly, but other protease inhibitors had virtually no effect. The inhibitory effect of aprotinin was specific for embryos at the blastula or gastrula stage. These results suggest that an aprotinin-sensitive protease involved in embryonic development is secreted from the embryos or appears on the surface of embryonic cells at these stages. We found that various serine proteases are in fact secreted from the embryos with their development and that some of them are sensitive to aprotinin.抄録全体を表示PDF形式でダウンロード (852K) -
Yasuhiro Takahashi, Minoru Nakamura1999 年 126 巻 5 号 p. 917-926
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーFe-S cluster, the nonheme-iron cofactor essential for the activity of many proteins, is incorporated into its target protein by an unknown mechanism. In Escherichia coli, genes in the ORF 1-ORF 2-iscS-iscU-iscA-hscB-hscA-fdx-ORF 3 cluster (the isc gene cluster) should be involved in the assembly of the Fe-S cluster since its coexpression with the reporter ferredoxin (Fd) dramatically increases the production of holoFd [Nakamura, M., Saeki, K., and Takahashi, Y. (1999) J. Biochem. 126, 10-18]. In this study we addressed the functional roles of the proteins encoded by the isc gene cluster with respect to the assembly of Fe-S clusters in four reporter Fds. Plasmids were constructed in which eight ORFs in the isc gene cluster were individually inactivated either by truncating the coding region or by introducing an oligonucleotide linker containing stop codons. By coexpressing these plasmids with reporter Fds, we show the iscS, iscA, hscA, and fdx genes to be required for the assembly of the Fe-S clusters. When these genes were absent from the coexpression plasmid, no overproduction was achieved in any reporter Fds examined. The inactivation of ORF 2 and hscB had a partial but appreciable effect on the production of some Fds. Deletion of ORF 1 produced no difference from the coexpression with the intact isc gene cluster. We also examined coexpression using the fdx gene in the isc gene cluster as a reporter Fd and identified iscS, hscB, hscA, and ORF 3 as being involved in the assembly of the [2 Fe-2 S] cluster in this protein. We propose a model in which the fdx gene product functions as an intermediate site for Fe-S cluster assembly.抄録全体を表示PDF形式でダウンロード (2404K) -
Yuko Nitahara, Yuri Aoyama, Tadao Horiuchi, Mitsuhide Noshiro, Yuzo Yo ...1999 年 126 巻 5 号 p. 927-933
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーSterol 14-demethylase P 450 (CYP 51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP 51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, O., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T., and Yoshida, Y. (1996) J. Biochem. 119, 926-933]. The cloned rat CYP 51 cDNA was expressed in Escherichia coli with modification of the N-terminal amino acid sequence, and the expressed protein (CYP 51m) was purified to gel-electrophoretic homogenity. The spectrophotometrically determined specific content of CYP 51m was 16 nmol/mg protein and the apparent molecular weight was estimated to be 53, 000 on SDS-PAGE. Soret peaks of the oxidized and reduced CO-complex of CYP 51m were observed at 417 and 447 nm, respectively. The purified CYP 51m catalyzed the 14-demethylation of lanosterol and 24, 25-dihydrolanosterol upon reconstitution with NADPH-P 450 reductase purified from rat liver microsomes. The apparent Km and Vmax values for lanosterol were 10.5 μM and 13.9 nmol/min/nmol P 450, respectively, and those for 24, 25-dihydrolanosterol were 20.0 μM and 20.0 nmol/min/nmol P 450, respectively. The lanosterol demethylase activity of the reconstituted system of CYP 51m was inhibited by ketoconazole, itraconazole and fluconazole with apparent IC50 values of 0.2, 0.7, and 160 μM, respectively.抄録全体を表示PDF形式でダウンロード (1524K) -
Keisuke Sakamoto, Tatsushi Mogi, Shunsuke Noguchi, Nobuhito Sone1999 年 126 巻 5 号 p. 934-939
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーTo probe the location of the quinol oxidation site and physical interactions for intersubunit electron transfer, we constructed and characterized two chimeric oxidases in which subunit II (CyoA) of cytochrome bo-type ubiquinol oxidase from Escherichia coli was replaced with the counterpart (CaaA) of caa3-type cytochrome c oxidase from thermophilic Bacillus PS 3. In pHNchi 5, the C-terminal hydrophilic domain except a connecting region as to transmembrane helix II of CyoA was replaced with the counterpart of CaaA, which carries the CuA site and cytochrome c domain. The resultant chimeric oxidase was detected immunochemically and spectroscopically, and the turnover numbers for Q1H2 (ubiquinol-1) and TMPD (N, N, N', N'-tetramethyl-p-phenylenediamine) oxidation were 28 and 8.5 s-1, respectively. In pHNchi 6, the chimeric oxidase was designed to carry a minimal region of the cupredoxin fold containing all the CuA ligands, and showed enzymatic activities of 65 and 5.1 s-1, and an expression level better than that of pHNchi 5. Kinetic analyses proved that the apparent lower turnover of the chimeric enzyme by pHNchi 6 was due to the higher Km of the enzyme for Q1H2 (220 μM) than that of cytochrome bo (48 μM), while in the enzyme by pHNchi 5, both substrate-binding and internal electron transfer were purturbed. These results suggest that the connecting region and the C-terminal α1-α2-β11-α3 domain of CyoA are involved in the quinol oxidation and/or physical interactions for inter-subunit electron transfer, supporting our previous proposal [Sato-Watanabe, M., Mogi, T., Miyoshi, H., and Anraku, Y. (1998) Biochemistry 37, 12744-12752]. The close relationship of E. coli quinol oxidases to cytochrome c oxidase of Gram-positive bacteria like Bacillus was also indicated.抄録全体を表示PDF形式でダウンロード (1279K) -
Kazuhisa Aoki, Ge-Hong Sun-Wada, Hiroaki Segawa, Shigemi Yoshioka, Nob ...1999 年 126 巻 5 号 p. 940-950
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーHuman UDP-galactose transporter (hUGT 1) and CMP-sialic acid transporter (hCST) are related Golgi proteins with eight putative transmembrane helices predicted by computer analysis. We constructed chimeric molecules in which segments of various lengths from the C- or N-terminus of hUGT 1 were replaced by corresponding portions of hCST. The chimeras were transiently expressed in UGT-deficient mutant Lec 8 cells, and their UGT activity was assessed by the binding of GS-II lectin to the transfected cells. The replacement of either the N- or C-terminal cytoplasmic segment by that of hCST did not affect the expression or activity of hUGT 1. A chimera in which the eighth helix and the C-terminal tail were replaced also retained the UGT activity, indicating that this helix is not involved in the determination of substrate specificity. In contrast, three types of chimeras, in which the first helix, the first and the second helices, and a segment from the seventh helix to the C-terminus were replaced, respectively, were expressed very infrequently in the transfected cells, and had no UGT activity. They are likely folded incorrectly and degraded by a quality-control system, since the amounts of their mRNAs were normal and the proteins were mainly localized in the ER. The first and the seventh helices are important for the stability of the transporter protein.抄録全体を表示PDF形式でダウンロード (4585K) -
Jun Hoseki, Takato Yano, Yoshinori Koyama, Seiki Kuramitsu, Hiroyuki K ...1999 年 126 巻 5 号 p. 951-956
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe whole-genome sequencing of an extreme thermophile, Thermus thermophilus, is now in progress. Like other genome projects, major concern is shifting from the sequence itself to post-sequencing research such as functional or structural genomics. Under such circumstances, the demand for convenient genetic-engineering tools is increasing. In this study we have increased the thermostability of a kanamycin-resistance gene product using strategies based on directed evolution in T. thermophilus to the upper limit of its growth temperature. The most thermostable mutant has 19 amino-acid substitutions, whereby the thermostability is increased by 20°C, but the enzymatic activity is not significantly changed. Most of the mutated residues are located on the surface of the protein molecule, and, interestingly, five of the 19 substitutions are those to proline residues. The evolved kanamycin-resistance gene products could be used as selection markers at the optimum growth temperature of T. thermophilus. The development of such a convenient genetic-engineering tool would facilitate post-sequencing research on T. thermophilus.抄録全体を表示PDF形式でダウンロード (1610K) -
Goro Kato, Shuichiro Maeda1999 年 126 巻 5 号 p. 957-961
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーc-Src is phosphorylated at specific serine and threonine residues during mitosis in fibroblastic and epithelial cells. These sites are phosphorylated in vitro by the mitotic kinase Cdk 1 (p 341cdc2). In contrast, c-Src in Y 79 human retinoblastoma cells, which are of neuronal origin, is phosphorylated at one of the mitotic sites, Ser 75, throughout the cell cycle. The identity of the serine kinase that nonmitotically phosphorylates c-Src on Ser 75 remains unknown. We now are able to show for the first time that Cdk 5 kinase, which has the same consensus sequence as the Cdk 1 and Cdk 2 kinases, is required for the phosphorylation in asynchronous Y 79 cells. The Ser 75 phosphorylation was inhibited in a dosedependent manner by butyrolactone I, a specific inhibitor of Cdk 5-type kinases. Three stable subclones that have almost no kinase activity were selected by transfection of an antisense Cdk 5-specific activator p 35 construct into Y 79 cells. The loss of the kinase activity caused an approximately 85% inhibition of the Ser 75 phosphorylation. These results present compelling evidence that Cdk 5/p 35 kinase is responsible for the novel phosphorylation of c-Sre at Ser 75 in neuronal cells, raising the intriguing possibility that c-Src acts as an effector of Cdk 5/p 35 kinase during neuronal development.抄録全体を表示PDF形式でダウンロード (2834K) -
Yukio Niimura, Michiko Tomori, Keiko Tadano-Aritomi, Toshihiko Toida, ...1999 年 126 巻 5 号 p. 962-968
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーTwo fractions of a major ganglioside from the kidney of the pacific salmon, Oncorhynchus keta, were eluted from a DEAE-Sephadex column in the monosialosyl fraction. The faster moving ganglioside (XI) on TLC was separated from the slower moving one (X 2) by HPLC using a silica beads column. By methylation analysis, chemical and enzymatic degradation, reaction with monoclonal antibodies, LSIMS, and 1H-NMR spectroscopy, X 1 was determined to be a monosialosyl ganglioside belonging to the ganglio-series with a unique Fucα1-3 GalNAc linkage at the nonreducing terminal:
Fucal-3 Gal NAcβ1-3 Galβ1-3 GalNAcβ1-4 [NeuAcα2-3] Galβ1-4 Glcβ1-1 Cer
Analysis of the lipophilic moiety indicated predominance of 24:1 fatty acid in combination with sphingenine. X 2 was found to have a glycon structure identical to X 1. The ceramide of X 2 consisted predominantly of saturated fatty acids (18:0 and 16:0). The tissue concentrations of X 1 and X 2 in kidney were 3.7 and 2.8 nmol/g, respectively.抄録全体を表示PDF形式でダウンロード (3102K) -
Jun Tamori, Nobuyuki Kanzawa, Takaho Tajima, Toru Tamiya, Takahide Tsu ...1999 年 126 巻 5 号 p. 969-974
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーA novel isoform of myosinase was purified to homogeneity from liver of spear squid by sequential chromatographies using SP Sephadex, hydroxylapatite, Zn/Co chelating affinity, and TSK-gel G 2000 SW columns. Myosinase activity was detected as a single peak of 45-kDa protein by gel filtration. The novel isoform of myosinase specifically hydrolyzed a rabbit skeletal muscle myosin heavy chain into products of 120 and 100 kDa in the presence of Co2+ ions, and the cleavage site in the myosin heavy chain was quite different from those of two known myosinase isoforms, I and II. Therefore, we named the novel isoform myosinase III. Myosinase III was also distinguishable from myosinase I by its amino-terminal sequence. The sequence showed similarity to an internal sequence of the astacin family.抄録全体を表示PDF形式でダウンロード (2843K)
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