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127 巻, 1 号
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Tohru Mizushima2000 年 127 巻 1 号 p. 1-7
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーDnaA protein, the initiator for chromosomal DNA replication in Escherichia coli, has various activities, such as oligomerization (DnaA-DnaA interaction), ATP-binding, ATPase activity and membrane-binding. Site-directed mutational analyses have revealed not only the amino acid residues that are essential for these activities but also the functions of these activities. Following is a summary of the functions and regulatory mechanisms of DnaA protein in the initiation of chromosomal DNA replication. ATPbound DnaA protein, but not other forms of the protein binds to the origin of DNA replication and forms oligomers to open-up the duplex DNA. This oligomerization is mediated by a DnaA-DnaA interaction through the N-terminal region of the protein. After initiation of DNA replication, the ATPase activity of DnaA protein is stimulated and DnaA protein is inactivated to the ADP-bound form to suppress the re-initiation of DNA replication. DnaA protein binds to acidic phospholipids through an ionic interaction between basic amino acid residues of the protein and acidic residues of phospholipids. This interaction seems to be involved in the re-activation of DnaA protein (from the ADP-bound form to the ATP-bound form) to initiate DNA replication after the appropriate interval.抄録全体を表示PDF形式でダウンロード (3622K) -
Mitsuaki Sugahara, Tsutomu Mikawa, Ryuichi Kato, Keiichi Fukuyama, Tak ...2000 年 127 巻 1 号 p. 9-11
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーMutM protein, which removes the oxidatively damaged DNA base product, 8-oxoguanine (GO), has been crystallized by means of a hanging-drop vapor-diffusion procedure using polyethyleneglycol monomethylether 2000 as a precipitant in 2-(cyclohexylamino) ethanesulfonic acid (CHES) buffer, pH9.8. The diffraction data derived from oscillation photographs indicate that the crystals belong to the monoclinic system and space group P21. The crystals have unit-cell dimensions of a=45.4 Å, b=62.0 Å, c=99.7 Å, and β=90.8°. Assuming that the asymmetric unit contains two molecules, the Vm, value was calculated to be 2.35 Å3•Da-1. The crystals diffracted X-rays to at least 2.1 Å resolution and were suitable for high-resolution X-ray crystal structure determination.抄録全体を表示PDF形式でダウンロード (1186K) -
Masaharu Kotani, Youichi Tojima, Yasushi Shimoda, Atsushi Irie, Hideo ...2000 年 127 巻 1 号 p. 13-22
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーTo evidence the notion that gangliosides involve neuronal cell interactions in the brain, we surveyed the presence of ganglioside-binding proteins in membrane lysates of adult rat cerebellum. Three proteins (p58, p90, and p160) were identified as GT1b-binding proteins by incubation of the blot of the membrane lysate with GT1b micelles. We generated a monoclonal antibody (mAb) specific to the polypeptide portion of the GT1b-binding proteins (YAK-2). The YAK-2 mAb specifically reacted with all three proteins on blots of proteins pretreated under nonreducing conditions for SDS-PAGE, but reacted mainly with p58 under reducing conditions, showing that p90 and p160 are oligomeric forms of p58. The binding activity of the YAK-2 mAb was completely inhibited by the presence of GT1b micelles, indicating the specificity of YAK-2 mAb for p58 and its oligomers. Immunohistochemical investigations revealed that both p58 and GT1b colocalize within the granular layer of adult rat cerebellum. Expression cloning of p58 cDNA was performed using YAK-2 mAb, and five putative clones were obtained. Among them, the nucleotide sequence of one cDNA completely matched that of rat brain-specific sodium-dependent inorganic phosphate cotransporter (rBNPI), a 61 kDa membrane protein. COS7 cells were transfected with a Flag-chimeric construct containing the rBNPI/p58 cDNA, and the membrane lysate was subjected to immunoprecipitation with anti-Flag antibody. One protein (64 kDa) was detected only with YAK-2 mAb, and the membrane lysate specifically bound to GT1b micelles. Taking together, we propose that rBNPI/p58 functions as a GT1b-binding protein in neuronal cells.抄録全体を表示PDF形式でダウンロード (3761K) -
Ki-Yong Kim, Inpyo Choi, Soung-Soo Kim2000 年 127 巻 1 号 p. 23-27
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーAn inhibitor of the proliferation of hepatic stellate cells (HSC) was purified from rat liver by a combination of gel filtration and ion exchange chromatography. The molecular mass of this non-arginase growth inhibitory factor (NAGIF) was determined to be 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The proliferation of HSC was inhibited by NAGIF with a 50% inhibitory dose of 5 nmol/liter. The inhibitory activity of NAGIF was not limited to HSC but also affected the growth of bovine endothelial cells and 3T6 fibroblasts. However, the growth of B 16 mouse melanoma was not inhibited by NAGIF. The NH2-terminal sequence of NAGIF, AEPVEPWS, is identical to an internal sequence of rat Zn-α2-glycoprotein. Although the action mode of this inhibitor remains to be investigated, it seems very likely that NAGIF is involved in the negative control mechanism of HSC growth.抄録全体を表示PDF形式でダウンロード (2955K) -
Masaaki Furuno, Ken Sato, Ikuro Kawagishi, Michio Homma2000 年 127 巻 1 号 p. 29-36
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF 60, PF 47, PF 45, PF 44, and PF 18, respectively), have been detected in polar flagella. PF 47 and PF 45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF 60. We found that PF 60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF 60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF 60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF 60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF 47 flagellin.抄録全体を表示PDF形式でダウンロード (4064K) -
An Easy Cell-Free Protein Synthesis System Dependent on the Addition of Crude Escherichia coli tRNAITakayuki Kanda, Kazuyuki Takai, Shigeyuki Yokoyama, Hiroshi Takaku2000 年 127 巻 1 号 p. 37-41
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe protein-synthesizing S 30 extract of Escherichia coil contains tRNA, which limits its applications in cell-free protein synthesis. Here, we show that at least Arg- and Seracceptor activities can be removed from a standard S 30 extract by treatment with an immobilized RNase A resin. This RNase-treated extract exhibits no protein synthesis activity, but regains it when supplied with crude E. coli tRNA and a small amount of human placental RNase inhibitor. The protein synthesis is dependent on the addition of tRNA in the presence of the RNase inhibitor. Chloramphenicol acetyltransferase was synthesized with this system and found to be active.抄録全体を表示PDF形式でダウンロード (2200K) -
Takashi Ohmoto, Kunihiko Moriyoshi, Kiyofiuni Sakai, Nobutake Hamada, ...2000 年 127 巻 1 号 p. 43-49
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーTwo trans-o-hydroxybenzylidenepyruvate hydratase-aldolases named tHBP HA A and tHBP HA B were purified from a cell-free extract of naphthalenesulfonate-assimilating Sphingomonas paucimobilis (formerly Pseudomonas sp.) TA-2 to an electrophoretically homogeneous state by successive column chromatographies on DEAE-cellulose, DEAE-Toyopearl 650 M, Sephacryl S-100, Hydroxyapatite, and Mono Q. These enzymes were similar to each other in molecular mass (ca. 37 kDa on SDS-PAGE, ca. 110 kDa on ultracentrifugation), thermal stability (<50°C) and optimum pH (pH 9.0). However, they differed from each other in N-terminal amino acid sequences, pH stability, Km values for trans-o-hydroxybenzylidenepyruvate (tHBP), and inhibition by p-chloromercuribenzoic acid (PCMB). tHBP HA B had a homologous N-terminal amino acid sequence with tHBP HAs from Pseudomonas vesicularis DSM 6383 (strain BN 6) and Sphingomonas aromaticivorans F 119, and tHBP HA A had a homologous sequence with tHBP HAs of Pseudomonas putida strain OUS 82, Pseudomonas sp. strain C 18 and NAH 7 plasmid. tHBP HA B was inhibited by PCMB, but tHBP HA A was not. Their Km values for tHBP were 9 and 3 μM, respectively. tHBP HA B was stable in the range of pH 7.1 to pH 10.7, and tHBP HA A was stable in the range of pH 6.0 to 9.3.抄録全体を表示PDF形式でダウンロード (1327K) -
Osamu Numata, Yasuhiro Kurasawa, Kohsuke Gonda, Yoshio Watanabe2000 年 127 巻 1 号 p. 51-56
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーTranslation elongation factor 1α (EF-1α) catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes. We previously reported that Tetrahymena EF-1α induced the formation of bundles of rabbit skeletal muscle filamentous actin (F-actin) as well as Tetrahymena F-actin [Kurasawa et al. (1996) Zool. Sci. (Tokyo) 13, 371-375], and that Ca2+/calmodulin (CaM) regulated the F-actin-bundling activity of EF-1α [Kurasawa et al. (1996) J. Biochem. 119, 791-798]. In the present study, we investigated the binding between Tetrahymena EF-1α and CaM using a Tetrahymena EF-1α affinity column, and the localization of EF-1α and CaM by indirect immunofluorescence. Only CaM in the Tetrahymena cell extract bound to Tetrahymena EF-1α in a Ca2+-dependent manner. In interphase Tetrahymena cells, EF-1α and CaM are colocalized in the crescent structure of the oral apparatus and the apical ring, while in dividing cells, they are colocalized in the division furrow. This is the first report describing the coexistence of EF-1α and CaM in the division furrow, suggesting that EF-1α and CaM are involved in the organization of contractile ring microfilaments during cytokinesis.抄録全体を表示PDF形式でダウンロード (3107K) -
Hiroko Ishida, Chika Yamashita, Yoshihiro Kuruta, Yuzo Yoshida, Mitsuh ...2000 年 127 巻 1 号 p. 57-64
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーSterol 12α-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid/chenodeoxycholic acid ratio in bile acid biosynthesis. The hepatic CYP8B level was elevated in streptozotocin-induced diabetic rats, and the elevated CYP8B was suppressed by insulin administration [Ishida, H. et al. (1999) J. Biochem. 126, 19-25]. The streptozotocininduced elevation of hepatic CYP8B mRNA concomitantly responded to the decrement of the serum insulin level. The CYP8B mRNA level in the cultivated rat hepatoma H4TG cells was strongly suppressed by insulin, although it was affected by dibutyryl cAMP or thyroxine to lesser extents. These observations demonstrate that CYP8B expression is dominantly regulated by the direct action of insulin on hepatocytes. A marked circadian rhythm (maximum at 13:00-16:00 and minimum at 1:00) was observed both on the mRNA level and the activity of CYP8B. This rhythm was shifted from that of cholesterol 7α-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis, showing a maximum at 22:00 and a minimum at 10:00, and this shift might oscillate the cholic acid/chenodeoxycholic acid ratio, which is increased in the late afternoon and decreased at midnight. The rhythm of CYP 8B was the inverse of the circadian variation of serum insulin level and was similar to the circadian rhythm of glucose 6-phosphatase. These facts and the potent suppressive effect of insulin on CYP 8B indicate that the oscillation of the serum insulin may be a factor in producing the circadian rhythm of CYP8B.抄録全体を表示PDF形式でダウンロード (3055K) -
Chiaki Sato, Jae-Hoon Kim, Yoshito Abe, Kazuki Saito, Shigeyuki Yokoya ...2000 年 127 巻 1 号 p. 65-72
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe extracellular domain of human EGF receptor (sEGFR) produced by CHO cells has been used in various biophysical studies to elucidate the molecular mechanism of EGFinduced receptor activation. We have found that the CHO sEGFR contains one oligosaccharide chain attached to an atypical N-glycosylation consensus sequence, Asn32-X33-Cys34. The oligosaccharide structure at Asn32 is a mixture of the monosialo and asialo forms of a core fucosylated biantennary complex-type oligosaccharide. Deletion of this atypical glycosylation site by replacement of Asn32 with lysine changed neither the expression nor function of the full length EGFR in CHO cells. The glycosylation at Asn32 in CHO sEGFR was incomplete: 20% of Asn32 remained unmodified. Thus, CHO sEGFR itself is heterogeneous with respect to the glycosylation at Asn32, which may cause problems in biophysical studies. An attempt to remove the oligosaccharide at Asn32 enzymatically did not succeed under nondenaturing conditions. Therefore, sEGFR with the mutation of Asn32→Lys32 is useful for biophysical and biochemical studies, and, particularly, for X-ray crystallography.抄録全体を表示PDF形式でダウンロード (2810K) -
Atsushi Watanabe, Izuru Yonemura, Kohsuke Gonda, Osamu Numata2000 年 127 巻 1 号 p. 85-94
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーTetrahymena F-actin-binding protein, which induces bundling of Tetrahymena F-actin, was localized to a division furrow during cytokinesis. We report here the cloning and characterization of the gene and cDNA of a Tetrahymena F-actin-binding protein. The cDNA encodes a protein comprising 579 deduced amino acids with a calculated molecular mass of 65.9 kDa. The predicted amino acid sequence shares 37.7, 41.8, and 39% identity with the sequences of yeast fimbrin, Arabidopsis thaliana fimbrin, and Dictyostelium discoideum plastin, respectively. The Tetrahymena F-actin-binding protein also shares two actin-binding domains previously identified in the fimbrin/plastin family, but lacks the EF-hand Ca2+-binding motif, suggesting that this protein is a novel-fimbrin-like protein in Tetrahymena. Moreover, we cloned a genomic DNA encoding the Tetrahymena fimbrin-like protein and performed Southern and Northern hybridizations. The results indicate that the genomic DNA possesses 9 introns and that both the gene and transcript of Tetrahymena fimbrin-like protein are single. Thus, we suggest that Tetrahymena fimbrin-like protein localizes to the division furrow and probably cross-links actin filaments in a Ca2+-insensitive manner during cytokinesis.抄録全体を表示PDF形式でダウンロード (3610K) -
Yinljo Soh, Marie W. Wooten2000 年 127 巻 1 号 p. 95-103
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーA protamine kinase from HL60 cells was purified to near homogeneity by DEAE-Sephacel, protamine-agarose, Hydroxylapatite, and S-200 chromatography. It was purified by 75.8-fold through four chromatographic steps, and 0.67% of total activity was recovered. The purified enzyme had an apparent molecular mass of 120 kDa and was activated by Mg2+ or Mn2+, but inhibited by Ca2+. Neither phospholipid nor phorbol ester significantly affected the enzyme activity. Staurosporine was the most potent inhibitor of the enzyme among the protein kinase inhibitors tested, K252a, H7, heparin, and staurosporine. The purified protamine kinase exhibited a maximum velocity of 5, 000 pmol/min/mg and Km of 1.3mM for protamine sulfate as a substrate. Myelin basic protein and protamine sulfate served as the best substrates for the protamine kinase among those tested. The activity of the protamine kinase remained unchanged upon treatment with PMA, retinoic acid, dimethyl sulfoxide, or 1, 25 dihydroxy vitamin D3, for 15min, while treatment with a differentiating agent, 1, 25 dihydroxy vitamin D3, for one week increased its activity. These results suggest that protamine kinase in HL60 cells is involved in the late stage of the macrophage-monocytic differentiation pathway and may play a role in maintenance of the differentiation after HL60 cells are committed.抄録全体を表示PDF形式でダウンロード (4291K) -
Xiaoqing Pan, Naoko Minegishi, Hideo Harigae, Hironori Yarnagiwa, Masa ...2000 年 127 巻 1 号 p. 105-112
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーTranscription factor GATA-2 is essential for the proper function of hematopoietic stem cells and progenitors. Two first exons/promoters have been found in the mouse GATA-2 gene, and a distal IS promoter shows activity specific to hematopoietic progenitors and neural tissues. To ascertain whether the two-promoter system is also utilized in the human GATA-2 gene, we isolated and analyzed a P1 phage clone containing this gene. The nucleotide sequence of the human GATA-2 gene 5' flanking region was determined over 10 kbp, and a human IS exon was identified in the locus through sequence comparison analysis with that of the mouse GATA-2 IS exon. RNA blotting and reverse-transcribed PCR analyses identified a transcript that starts from the IS exon in human leukemia-derived cell lines. The IS-originated transcript was also identified in CD34-positive bone marrow and cord blood mononuclear cells, which are recognized as clinically important hematopoietic stem cell-enriched fractions. Phylogenic comparison of the human and mouse GATA-2 gene sequences revealed several regions in the locus that exhibit high sequence similarity. These results demonstrate that the GATA-2 gene regulatory machinery is conserved among vertebrates. The fact that the human IS promoter is active in the hematopoietic stem cell/progenitor fraction may be an important clue for the design of a vector system that can specifically express various genes in hematopoietic stem cells and progenitors.抄録全体を表示PDF形式でダウンロード (2802K) -
Hiroyuki Nakagawa, Masaru Yoshida, Shigeaki Miyamoto2000 年 127 巻 1 号 p. 113-119
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーNitric oxide (NO) acts as a cytostatic agent to induce neuronal differentiation of PC12 cells after nerve growth factor (NGF) treatment. We newly subcloned PC12K cells that extended neurites after depolarization with high KC1. Here we present evidence that the neuronal differentiation of PC12K cells caused by depolarization with high KC1 is mediated by endogenous NO. The outgrowth of neurites was significantly inhibited by 2mM N-nitro-L-arginine methyl ester (L-NIV1AE), and 10mM L-NAME was necessary for complete inhibition. The inhibition of NGF-dependent neurite outgrowth by L-NAME was abolished by depolarization of cells with KCl. The expression of neuronal- and endothelial-NO-synthase in PC12K cells was confirmed by immuno-cytochemical and immuno-blotting analyses with the respective monoclonal antibodies. However, the expression of inducible-NO synthase was not observed in PC12K cells cultured with high KC1 under the depolarization conditions with 45mM KCI. We observed the increase of NO in the differentiated PC12K cells using diaminofluorescein, a novel fluorescent indicator for NO.抄録全体を表示PDF形式でダウンロード (4464K) -
Masayo Kojima, Kazuya Oguro, Keiko Sawabe, Yoshiko Iida, Rie Ikeda, At ...2000 年 127 巻 1 号 p. 121-127
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーPreviously we demonstrated that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by ATP-dependent proteolysis in serotonin producing mast cells [Hasegawa et al. (1995) FEBS Lett. 368, 151-154]. We searched for the major proteases involved in the rapid degradation of TPH in RBL2H3 cells. Among various protease inhibitors tested, proteasome inhibitors MG115, MG101, MG132, and lactacystin effectively inhibited the intracellular degradation of TPH. Administration of the proteasome inhibitors to cultured cells caused more than a 5-fold accumulation of TPH. Administration of the inhibitors together with cycloheximide stabilized the amount of TPH with no appreciable increase or decrease. Although MG-series proteasome inhibitors could inhibit calpain, the involvement of calpain was excluded since the cysteine protease inhibitor E-64-d, which acts on calpain, had no effect. Extracts of RBL2H3 cells were shown to contain a protease that digests TPH in an ATP-dependent manner and is sensitive to proteasome inhibitors. The ubiquitination of TPH could be visualized by Western blot analysis using both anti-TPH and anti-ubiquitin antibodies. Based on these results, we conclude that 26S proteasomes are mainly involved in the degradation of TPH. In the reported amino acid sequences of TPH from various sources including human, rabbit, rat, and mouse, a PEST sequence that is widely shared among short-lived proteins has been recognized. It was noted that the PEST sequence lies within the most conserved portion of the enzyme, the pteridine binding site.抄録全体を表示PDF形式でダウンロード (2591K) -
Kazuo Yamamoto, Yukiko Konami, Toshiaki Osawa2000 年 127 巻 1 号 p. 129-135
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーLectins are carbohydrate-binding proteins widely used in biochemical, inununochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-I43 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galβ1-3GaINAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manal-6-(Manαl-3)Manal-6 Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies.抄録全体を表示PDF形式でダウンロード (1245K) -
Kazuo Yamamoto, Ichiro N. Maruyarna, Toshiaki Osawa2000 年 127 巻 1 号 p. 137-142
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーBauhinia purpurea lectin (BPA) is one of the β-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called “cyborg lectins”) with distinct and desired carbohydrate-binding specificities.抄録全体を表示PDF形式でダウンロード (1516K) -
Hiroki Tsuruta, Yasuo Aizono2000 年 127 巻 1 号 p. 143-149
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーPsychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3. 1. 3. 48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2, 004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of proteinserineithreonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4 B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity.抄録全体を表示PDF形式でダウンロード (2390K) -
Hiroyasu Furukawa, Tatsuya Haga2000 年 127 巻 1 号 p. 151-161
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe M2 muscarinic acetylcholine receptor mutant (M, mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein NEP) at its N-terminus and expressed in Escherichia coli. The expression level was 0.2 urnol receptor per 100 ml culture, as assessed as [3H] L-quinuclidinyl benzilate ([3H] QNB) binding activity, when the BL 21 strain was cultured at 37°C to a late growth phase and the expression was induced by isopropyl β-thiogalactoside at 20°C. No [3H]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37°C instead of 20°C. The MBP-M2 mutant expressed in E. coli showed the same ligand binding activity as the M2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [3H] QNB with carbamylcholine and atropine. The MBP M2 mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein Go or Gil in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [35S] GTPγS binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [35S]GTPγS binding were the same as those observed for the M2, mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol. These results indicate that the MBP-M2 mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M2 muscarinic receptor.抄録全体を表示PDF形式でダウンロード (1955K) -
Michael Lehnerer, Johannes Schulze, Klaus Achterhold, David F. V. Lewi ...2000 年 127 巻 1 号 p. 163-169
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーA cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R). Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4 (Δ2-27) lacking the NH2-terminal signal anchor sequence. The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis. Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system. Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Δ2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6.1-fold increase in the Km values with unchanged Vmax; this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NADPH consumption and H2O2 formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through π-stacking with Phe-296 located close to the heme unit. All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R.抄録全体を表示PDF形式でダウンロード (791K)
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