巻号一覧
120 巻, 5 号
選択された号の論文の27件中1～27を表示しています
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Sayoko Ihara, Akihiro Iwamatsu, Toshihide Fujiyoshi, Akiko Komi, Takao ...1996 年 120 巻 5 号 p. 865-868
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe have screened for a factor that induces neurite outgrowth by PC 12 cells only after NGF pretreatment in the supernatants of 38 transformed cell lines, finding three positive clones. The factor showing the strongest activity was purified and identified as IL 6. In this NGFIL 6 system, the process of neurite outgrowth by PC 12 cells can be separated into NGF-dependent and IL6-dependent steps. The IL6-dependent step requires RNA synthesis, suggesting that IL 6 induces new gene expression depending on NGF-priming. These findings suggest that the gene expression during the differentiation process is regulated by at least two signals.抄録全体を表示PDF形式でダウンロード (1569K) -
Kikuo Ogata, Rie Ohno, Sakura Morioka, Kazuo Terao1996 年 120 巻 5 号 p. 869-880
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーTo obtain direct evidence for the attachment of 5SrRNA-ribosomal L 5 protein particles (5 SRNP) and methionine-tRNA (tRNAmet) to methionyl-tRNA synthetase (MetRS) in the macromolecular aminoacyl-tRNA synthetase (ARS) complex of rat liver, a MetRS-5SRNP-tRNAmet complex was dissociated from the macromolecular ARS complex fraction by n-octyl-β-D-glucoside (Method I) or by ω-aminooctyl agarose (Method II) chromatography. The dissociated MetRS complex fraction was purified by gel filtration followed by tRNASepharose chromatography using partially purified tRNAmet in Method I, and by hydrophobic interaction chromatography in Method II. In both methods, final SuperdexTM200 chromatography showed that MetRS activity was present in the region corresponding to the molecular weight of the MetRS-5SRNP-tRNAmet complex (Mr 200, 000). One main protein band corresponding to the molecular weight of MetRS was observed on SDS-PAGE of the final product, which was concentrated by lyophilizing after dialysis against water. Using serum albumin as an inhibitor of adhesion of L 5 to the microconcentrators which was used to concentrate the final product, a distinct L 5 band was detected on SDS-PAGE, the intensity of which was comparable to that of the MetRS band. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 5 SrRNA. Dot blot analysis using an antibody against ribosomal protein L 5 showed that L 5 was present in the SuperdexTM200 fractions prepared by both methods. The MetRS specific activities in MetRS complex fractions incubated without tRNA increased during the purification procedures, indicating that endogeneous tRNAmet exists stably in the MetRS complex. 5 SRNP and 5 SrRNA markedly enhanced the MetRS activity in the MetRS complex, indicating that 5 SRNP(A) plays a role as a positive effector of MetRS.抄録全体を表示PDF形式でダウンロード (2577K) -
Bo Zhou, Guo-Zhong Jing1996 年 120 巻 5 号 p. 881-888
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe conformational and activity changes of a family of peptide fragments of staphylococcal nuclease R, which extend from residues -6 to 102, -6 to 110, -6 to 121, -6 to 135, and -6 to 141, during unfolding and refolding in different concentrations of guanidine hydrochloride have been studied. The studies indicate that the conformational stability in guanidine hydrochloride solution of the N-terminal fragment increases with increasing chain length, and that interaction and recognition between amino acid residues which are related to formation of the native conformation also increase with growth of the peptide chain, but such interaction becomes effective only when the polypeptide chain reaches a certain length. The changes in conformation and catalytic activity of the N-terminal fragments during unfolding and refolding demonstrate that conformational adjustments are necessary during chain elongation to generate the native conformation of a biologically active protein.抄録全体を表示PDF形式でダウンロード (848K) -
Koichi Nakazato, Masatoshi Muraoka, Eijiro Adachi, Toshihiko Hayashi1996 年 120 巻 5 号 p. 889-894
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーIt is known that the type IV collagen extracted from EHS tumor assembles under a physiological condition, but not in a gel form. The EHS type IV collagen requires the other basement membrane components, lamininl, heparansulfate proteoglycan, and/or nidogen for gelation. On the other hand, Muraoka et al. reported that the bovine lens capsule type IV collagen alone gelated under a unique and unexpected condition of 2M guanidine-HC1 and 50mM dithiothreitol, a condition which is thought to be dissociative for most biological macromolecules, including extracellular matrix [Muraoka, M. et al. (1996) J. Biochem. 119, 167-172]. The present report shows that the bovine lens capsule type IV collagen formed a gel under physiological conditions of pH and ionic environment, though the apparent rigidity of the gel was weaker than that of the gel formed in 2M guanidine-HCI and dithiothreitol. The rigidity depended greatly on the incubation temperature and NaCl concentration of the type IV collagen solution, as observed in terms of the contractility of gel volume under centrifugal force. The gel formed in 150mM NaCl and 20mM phosphate, pH 7.3, at 28°C contracted to 20% of the original volume on centrifugation of 1, 800×g for 10min, while the gel formed at 4°C, where type I collagen did not gelate at all, retained 90% of the original volume at the same centrifugal force. NaCl concentration was another impor-tant factor influencing the mechanical properties of type IV collagen gel. The gel formed at 150mM showed maximal rigidity in the range of 0 to 300mM in terms of the contractility on centrifugation. An image of a Pt/C replica of the gelated type IV collagen reconstituted at 4 or 28°C in 20mM phosphate, pH 7.3, containing 150mM NaCl showed fine meshworks consisting of rather homogeneous pore sizes, resembling the skeletal structure of basal lamina. Since the condition where the type IV collagen alone formed gels was physiological in terms of ionic strength and pH, the aggregate structure and gel properties might reflect the in vivo type IV collagen supramolecular structure and the property.抄録全体を表示PDF形式でダウンロード (3535K) -
Takao Ohyashiki, Tomokazu Karino, Satomi Suzuki, Katsuhiko Matsui1996 年 120 巻 5 号 p. 895-900
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe effects of Al3+ on Fe2+-induced lipid peroxidation in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) were examined under acidic conditions. The stimulatory effect of Al3+ on Fe2+-induced lipid peroxidation in the liposomes showed a biphasic response against pH variation, and the maximum stimulation was observed around pH 6.0. In addition, it was found that the stimulatory effect of Al3+ on the lipid peroxidation was dependent on the proportion of PS in the liposomes. On the other hand, the lipid peroxidation in PC liposomes was not stimulated by the addition of Al3+. From these findings, it is suggested that the Al3+ effect on Fe2+-induced lipid peroxidation under acidic conditions is largely dependent on the phospholipid composition. Trivalent cations such as Tb3+ and Ga3+ also stimulated Fe2+-induced lipid peroxidation in PC/PS liposomes under acidic conditions, but divalent cations (Zn2+ and Mn2+) showed no stimulatory effect. The extents of Fell disappearance and Fe3+ formation during the reaction were enhanced by the addition of Al3+ or Ga2+, but Tb3+ had no effect on Fe2+ disappearance. The results with 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) showed that the fluorescence anisotropy of DPH-labeled PC/PS liposomes under acidic conditions was increased by the addition of Al3+. Furthermore, there is a relation between the extents of the fluorescence anisotropy of the complex and TBARS production. In contrast, the fluorescence anisotropy of DPH molecules embedded in PC liposomes was not changed by the addition of Al3+. Based on these results, a possible mechanism of the stimulatory effect of Al3+ on Fe2+-induced lipid peroxidation under acidic conditions is discussed.抄録全体を表示PDF形式でダウンロード (721K) -
Yasushi Hasegawa, Tomoaki Kikuta, Yoh Okamoto1996 年 120 巻 5 号 p. 901-907
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーA complex of 110-kDa heavy chain and calmodulin was isolated from porcine aorta media smooth muscle and identified as myosin I. The isolated myosin I consisted of equimolar amounts of 110-kDa heavy chain and calmodulin. The addition of exogenous calmodulin to the complex revealed that a maximum of two molecules of calmodulin could be bound to the heavy chain. Isolated complex bound to F-actin in an ATP-dependent manner and its Mg2+-ATPase activity was activated by F-actin. In addition, it bound to phospholipid, which is a characteristic property of myosin I. Calcium ions induced a structural change, which was revealed by a difference in the cleavage pattern and for rate of cleavage by α-chymotrypsin. This behavior was similar to that reported for brush border myosin I [L. M. Collins and A. Bretscher (1988) J. Cell. Biol. 106, 367-373]. Calcium-dependent structural change of a complex of 110-kDa heavy chain and calmodulin was found from its solubility change at various NaCl concentrations in the presence of ATP. A complex of 116-kDa heavy chain and calmodulin, possibly another type of myosin I, was also isolated. A polyclonal antibody against the complex of 110-kDa heavy chain and calmodulin did not recognize the 116-kDa heavy chain. This result suggests that at least two types of myosin Is may exist at the protein level in porcine aorta media smooth muscle.抄録全体を表示PDF形式でダウンロード (2137K) -
Tetsuro Sato, Katsuya Baba, Yoshiaki Takahashi, Toshio Uchiumi, Shoji ...1996 年 120 巻 5 号 p. 908-914
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーFatty acid-binding protein (FABP) has been isolated from rat liver cytosol by two steps of gel-permeation chromatography on Sephadex G-75 and Sephacryl S-100 after ammonium sulfate precipitation. FABP fraction was eluted as two well-separated peaks, fractions A and B, by reversed-phase high-performance liquid chromatography (HPLC). The structural difference between the two fractions was investigated by lysyl endopeptidase digestion followed by reversed-phase HPLC of the digests, which identified a peptide corresponding to residues 58 through 78 as the modified peptide. Matrix-assisted laserdesorption-ionization mass spectrometry and other chemical analyses of the peptides established the modification in fraction A as cysteine-thiolation at cysteine-69. This was confirmed by reduction and reoxidation of the peptide and the parent molecules. The modification did not affect binding of fluorescent derivatives of fatty acids. However, the modified species was more susceptible to proteolysis by bovine spleen cathepsin B and cathepsin D than the unmodified species. The presence of a relatively large amount of cysteine (but not of glutathione) mixed-disulfide form of FABP suggests some physiological role of this modification related to the redox status of the cell [Thomas, J. A., Poland, B., and Honzatko, R. (1995) Arch. Biochem. Biophys. 319, 1-9], and accounts, at least in part, for the extensive heterogeneity of liver FABP.抄録全体を表示PDF形式でダウンロード (816K) -
Akeo Shinkai, Akiko Hirano, Kazuo Aisaka1996 年 120 巻 5 号 p. 915-921
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe lipase gene from Pseudomonas aeruginosa was randomly mutated by error-prone PCR to obtain thermostable mutants, followed by screening for thermostable mutant lipases. Out of about 2, 600 transformants, four thermostable clones were obtained. Their nucleotide sequences showed that they had two or three amino acid substitutions. Analysis of the thermal stabilization of these mutant lipases indicated that Asn-163 to Ser and Leu-264 to Pro mutations were essential for the increased stability of the lipase. We expressed a mutant lipase (StLipA-5) having only the Asn-163 to Ser mutation and another (StLipA-6) having only the Leu-264 to Pro mutation in P. aeruginosa PAO 1161, purified them, and then confirmed that the temperature which causes a 50% decrease in the activity of the non-treated enzyme on treatment for 30 min was increased by 1.5 and 3°C, respectively, compared to the wild-type enzyme. However, the thermal stability of the mutant lipase (StLipA-7) having both mutations was increased only by 2.5°C. These mutant lipases were stabilized through a decrease in activation entropy. Kinetic studies showed that the kcat/Km values of StLipA-5, StLipA-6, and StLipA-7 were decreased by 14.4, 52.9, and 26.0%, respectively. Interestingly, the pH-stabilities of StLipA-6 and StLipA-7 were also increased, especially at alkaline pH. Based on these results, the tertiary structure and mechanism of stabilization of the lipase were discussed.抄録全体を表示PDF形式でダウンロード (1797K) -
Takayuki Tobita, Fumiko Hiraide, Jun-Ichi Miyazaki, Tadashi Ishimoda-T ...1996 年 120 巻 5 号 p. 922-928
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーTropomyosin isoforms in eggs of several species of sea urchins are classified into two types, muscle-type and nonmuscle-type, based on their antigenicities. Their actin-binding abilities were investigated using muscle-type isoform (32 K) and nonmuscle-type isoform (30 K), which were purified by the method previously reported and separated by isoelectric focusing from eggs of sea urchin, Strongylocentrotus intermedius. Co-sedimentation assays revealed that 32 K could stoichiometrically bind to actin filaments independently of the 30 K, but 30 K alone bound very poorly. The actin-binding of 30 K was, however, considerably increased in the presence of 32 K, and the molar ratio of the bound 30 K and 32 K was approximately 1:1. The increase in the actin-binding of 30 K is probably caused by the interaction of 30 K with 32 K in a head-to-tail manner, as indicated by the higher specific viscosity of the mixture than that of 32 K alone.抄録全体を表示PDF形式でダウンロード (6902K) -
Seiichi Mizushima, Hironori Sato, Takaaki Negishi, Hiroko Koushima, At ...1996 年 120 巻 5 号 p. 929-933
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーProstacyclin-stimulating factor (PSF) is a protein which acts on vascular endothelial cells and stimulates the production of prostacyclin. Recently, we were able to purify PSF from the conditioned medium of cultured human diploid fibroblasts and clone PSF cDNA. In this study, we screened a human genomic library and isolated genomic clones to determine the structure of the human chromosomal PSF gene. By determining the nucleotide sequence and transcription initiation site of this gene, we found that it comprises 5 exons and 4 introns. Southern hybridization analysis indicated the presence of a single copy of the PSF gene per haploid set of chromosomes. The 300 by upstream of the transcription initiation site had a very high GC content, and 7 binding sites for the transcription regulating factor Sp 1 were present.抄録全体を表示PDF形式でダウンロード (1629K) -
Kazunori Mizuno, Toshihiko Hayashi1996 年 120 巻 5 号 p. 934-939
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe heparin affinities of heat-treated type V collagen α-chains and the triple-helical molecules were evaluated in terms of the NaCl concentration required for prevention of binding to a heparin-Sepharose column. After heat treatment, α1 (V) chain required approximately two-fold higher NaCl concentration to pass through the column than the other two chains, α2 (V) and α3 (V). Thus, the heparin affinity of α1 (V) may be approximately two-fold higher than those of the other α (V)-chains. The type V collagen molecules in triple-helical conformation were separated into two fractions at 170mM NaCl in 20mM phosphate buffer, pH 7.2, containing 2M urea; bound and non-bound. The ratio of the three α-chains, α1 (V): α2 (V): α3 (V) was 2:1:0 and 1:1:1 in the bound and flow-through fractions, respectively, on analysis by SDS-PAGE. The differential affinity of the two fractions could be accounted for by the number of α1 (V) chains in the triple-helical molecule, if these fractions contained triple-helical subtypes with the chain compositions of [α1 (V)]2α2 (V) and α1 (V)α2 (V)α3 (V), respectively. From the comparison of the NaCl concentration required for prevention of the binding, [α1 (V)]2α2 (V) had about two-fold higher affinity than α1 (V)α2 (V)α3 (V), and the separated α1 (V) chain showed an intermediate affinity. A possible explanation for difference in heparin affinity among the subtypes of molecules and the separated α-chains is that the heparin affinity of type V collagen molecule is governed by the number of α1 (V) chains contained in the molecule and that steric restraint in a triple-helical conformation weakens the binding of al(V) chain to heparin.抄録全体を表示PDF形式でダウンロード (2698K) -
Eiro Muneyuki, Toru Hisabori, Takeshi Sasayama, Katsura Mochizuki, Mas ...1996 年 120 巻 5 号 p. 940-945
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe interactions of substoichiometric TNP-ATP and F1-ATPase from Escherichia coli (EF1) were examined and compared with those in the case of mitochondrial F1-ATPase (MF1) and F1-ATPase from thermophilic Bacillus PS 3 (TF1). EF1 hydrolyzed substoichio-metric TNP-ATP faster than TF1 or MF1, although some 20% of the TNP-ATP remained unhydrolyzed even in the presence of excess chase ATP. The affinity of the catalytic site of EF1 for the product, TNP-ADP, was weaker than that of TF1 or MF1, and the TNP-ADP was readily released upon addition of excess ATP. The amplitude of the difference absorption spectrum induced by binding of TNP-AT(D)P to EF1 was smaller than that of MF1 or TF1 under similar experimental conditions. When an excess amount of TNP-ATP was added to EF1 and the change of the difference spectrum was measured, the shape of the difference spectrum of the ATP-replaceable fraction was very similar to that in the case of binding of TNP-ATP to the isolated β subunit of TF1, indicating that the rapidly replaceable fraction of bound TNP-ATP was actually at the catalytic site and most of the non-replaceable portion was bound at noncatalytic sites. Weaker affinity of the catalytic site for TNP-ATP may account for the heterogeneous binding and hydrolysis under the conditions described in this paper.抄録全体を表示PDF形式でダウンロード (747K) -
Eiji Ohmae, Toshiyuki Kurumiya, Shio Makino, Kunihiko Gekko1996 年 120 巻 5 号 p. 946-953
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe acid and thermal unfolding of Escherichia coli dihydrofolate reductase (DHFR) were studied by means of circular dichroism (CD) and fluorescence spectroscopy. There existed at least one intermediate around pH 4 in the acid unfolding process at 15°C, in which the tertiary structure was disrupted before unfolding of the secondary structure. The fluorescence energy transfer from intrinsic tryptophan residues to 1-anilinonaphthalene-8-sulfonate suggested the disruption of the tertiary structure around some tryptophan residues of the intermediate. The thermal unfolding process at pH 7.0 also involved at least one intermediate having a disrupted tertiary structure and a folded secondary structure. The three-state thermodynamic analysis showed that the intermediate in thermal unfolding was less stable by 1.8 kcal f mol than the native state. The similarity of the far-ultraviolet CD spectra of acid and thermally unfolded forms suggests that both types of unfolding produce the same structure, which may be a molten globule intermediate such as that in the folding kinetics of DHFR. The acid and thermal unfolding were depressed in the presence of KCl due to stabilization of the native form.抄録全体を表示PDF形式でダウンロード (1002K) -
Atsuko Yoneda, Kyoko Kojima, Isamu Matsumoto, Kazuo Yamamoto, Haruko O ...1996 年 120 巻 5 号 p. 954-960
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーVitronectin is a multifunctional glycoprotein regulating the fibrinolysis, complement, and coagulation systems in plasma, besides exhibiting cell-spreading activity. Porcine vitronectin has an unusually small molecular mass among the vitronectins hitherto found, which seems to make it hard for it to retain all the known activities. In this study, the complete primary structure of porcine vitronectin was elucidated by cloned cDNA and glycoprotein analyses. A coding sequence of 459 amino acids including a signal peptide of 19 amino acids was deduced from the cDNA. The coding sequence showed 70.3% homology with that of human vitronectin, but porcine vitronectin lacked 22 amino acids in the connecting region. One amino acid substitution resulted in the loss of a potential glycosylation site in accordance with the finding on glycopeptide analyses that porcine vitronectin contained two kinds of glycosylated sequences, while human vitronectin contained three. C-Terminal analysis of porcine vitronectin indicated that an 80 amino acid fragment was completely removed from the C-terminal end on proteolytic processing. Thus, porcine vitronectin only exists in a truncated single-chain form representing the most compact functional form of vitronectin, which suggests the lack of functional necessity of the truncated C-terminal fragment.抄録全体を表示PDF形式でダウンロード (849K) -
Shunsuke Aoki, Kuniaki Takahashi, Kunio Matsumoto, Toshikazu Nakamura1996 年 120 巻 5 号 p. 961-968
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーHepatocyte growth factor (HGF), a ligand for c-Met receptor tyrosine kinase, regulates the cell growth, migration and morphogenesis of a wide variety of cells, and plays important roles in organogenesis in embryos as well as regeneration of organs. HGF is structurally conserved in Xenopus laevis, and the Xenopus HGF gene is expressed in neurula and tailbud embryos. In the present work, we cloned complementary DNA for the Xenopus Met/HGF receptor and analyzed its expression during embryogenesis. The open reading frame of Xenopus c-met is 4, 125 base pairs long and encodes a putative polypeptide of 1, 375 amino acids. The deduced amino acid sequences of the Xenopus and human Met proteins are 63% identical, the tyrosine kinase domains in the intracellular regions showing particular homology, over 90% identity in the amino acid sequences between Xenopus and human. During Xenopus embryogenesis, c-met mRNA was expressed at a high level from the gastrula to the neurula stage, while HGF mRNA expression was seen from the early neurula stage. Whole mount in situ hybridization showed that c-met mRNA was specifically localized in the foregut region, mesenchymal tissue of the tailbud, and neural tissues in neurula embryos. Thus, c-Met is a highly conserved molecule in a wide range of species, as is its ligand, HGF. The HGF-Met system may be involved in early multiple organogenesis in Xenopus embryos.抄録全体を表示PDF形式でダウンロード (4642K) -
Yasuji Koyama, Toshio Ichikawa, Eiichi Nakano1996 年 120 巻 5 号 p. 969-973
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーA urate oxidase (uricase) gene was cloned from Candida utilis with an oligonucleotide probe based on the amino acid sequence of cyanogen bromide-cleaved uricase. The uricase gene contains 909 base pairs and encodes a protein with a predicted mass of 34, 193 Da. Candida uricase was similar (49% match in amino acid sequence) to the uricase from Aspergillus flavus. The uricase from Candida utilis has four cysteines and one of them, Cys168, participates in the enzyme activity. This enzyme was expressed to a level of about 20% of total cellular protein in an Escherichia coli cell as a soluble and functional form.抄録全体を表示PDF形式でダウンロード (1631K) -
Takahiro Shintani, Mizue Kobayashi, Eiji Ichishima1996 年 120 巻 5 号 p. 974-981
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe structural determinants of S1 substrate specificity of aspergillopepsin I (API; EC 3. 4. 23. 18), an aspartic proteinase from Aspergillus saitoi, were investigated by site-directed mutagenesis. Aspartic proteinases generally favor hydrophobic amino acids at P1 and P1'. However, API accommodates a Lys residue at P1, which leads to activation of trypsinogen. On the basis of amino acid sequence alignments of aspartic proteinases, Asp-76 and Ser-78 of API are conserved only in fungal enzymes with the ability to activate trypsinogen, and are located in the active-site flap. Site-directed mutants (D76N, D76E, D76S, D76T, S78A, and ΔS78) were constructed, overexpressed in Escherichia coli cells and purified for comparative studies using natural and synthetic substrates. Substitution of Asp-76 to Ser or Thr and deletion of Ser-78, corresponding to the mammalian aspartic proteinases, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at P1. In contrast, substrates with a hydrophobic residue at P1 were effectively hydrolyzed by each mutant enzyme. These results demonstrate that Asp-76 and Ser-78 residues on the active site flap play important roles in the recognition of a basic amino acid residue at the P1 position.抄録全体を表示PDF形式でダウンロード (3998K) -
Yuri Aoyama, Tadao Horiuchi, Yuzo Yoshida1996 年 120 巻 5 号 p. 982-986
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe occurrence of sterol 14-demethylase in rat brain microsomes was confirmed. The brain microsomes from adult rats converted lanosterol into its 14-demethylated products, 4, 4-dimethylcholesta-8, 24-dienol, and 4, 4-dimethylcholesta-8, 14, 24-trienol, in the presence of NADPH and molecular oxygen. This metabolism of lanosterol was inhibited by carbon monoxide and ketoconazole, a potent inhibitor of sterol 14-demethylase P450 (P45014DM or CYP51). These facts indicated the occurrence of lanosterol 14-demethylation in rat brain microsomes and its dependency on P45014DM. A representative value of the lanosterol demethylase activity of the brain microsomes was 8.4 pmol/min/mg protein or 640 pmol/min/nmol of total P450. The former was about one-thirteenth of the corresponding value observed with liver microsomes from the same rats, while the latter was 4-times higher than the corresponding value obtained with the liver microsomes. This fact suggested that the ratio of P45014DM to other P450 species was higher in brain than in liver. Lanosterol 14-demethylation is situated at the root of the sterol-biosynthetic branch of the mevalonic acid pathway. Therefore, the present finding enzymologically supports the existence of the sterol biosynthetic pathway in brain.抄録全体を表示PDF形式でダウンロード (620K) -
Midori Nomura, Zhihua Zou, Tadashi Joh, Yoshihiro Takihara, Yoichi Mat ...1996 年 120 巻 5 号 p. 987-995
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーRaelα, Raelβ, and Raelγ cDNAs isolated from retinoic acid-treated mouse embryonal carcinoma F9 cells encode cell surface proteins sharing partial homology with MHC class I molecules, and mRNAs corresponding to these cDNAs were detected exclusively in early mouse embryos, especially in the head region. To initiate studies on their roles, the rae1α gene and the genomic DNAs covering the complete coding regions of the rae1β and rae1γ genes were isolated and their structures were analyzed. Although the coding regions of the three rae1 genes were highly homologous, the restriction map of the 5'-end region of the rae1α gene differed from that of the rae1β and rae1γ genes. The rae1 family members were mapped by FISH on mouse chromosome 10A4 region. Genomic DNAs hybridizable with a Rae1 cDNA were not detected in rat and human. Rae1 genes were preferentially expressed in early mouse embryos, preferentially in the brain, and RAE1 proteins were anchored on the cell surface by a glycosyl phosphatidylinositol (GPI)-tail, a feature shared by important cell surface ligands.抄録全体を表示PDF形式でダウンロード (4431K) -
Koji Nakamura, Toshihide Nukada, Kohbun Imai, Hiroyuki Sugiyama1996 年 120 巻 5 号 p. 996-1001
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe α subunits of Gq family G proteins, GL1α and GL2α, are bovine homologues of mouse G14α and G11α, respectively, and are closely related to each other. When expressed in Xenopus oocytes together with metabotropic glutamate receptors, GL2α activates endogenous phospholipase C (PLCx) in response to glutamate stimulation, whereas GL1α inhibits the activation of PLCx. By examining the properties of 10 chimeras between GL1α and GL2α and their mutants, we tried to identify the regions on the Gα proteins that are important for the activation of PLCx. The results indicated that a necessary (but not sufficient) condition for a chimeric Gα protein to be able to clearly activate PLCx was that its N-terminal quarter portion should be derived from GL2α. No correlation was found between the origin (GL1α or GL2α) of C-terminal regions of the chimeras and the ability of chimeras to activate PLCx. One of the chimeras is different from GL2α at only four amino acid residues in the N-terminal region, and yet it could not activate PLCx. When one of the four residues, Ser-59, in the chimera was mutated back to Ala as in the original GL2α, the resulting mutant became capable of activating PLCx. This residue is localized in the midst of the N-terminal linker connecting the two major domains in the Gα proteins. These results indicate that Ala-59 is critical for the activation of PLCx, and that the linker may play important roles in determining functions of Gα proteins.抄録全体を表示PDF形式でダウンロード (797K) -
Satsuki Minamida, Kyoko Aoki, Shunji Natsuka, Kaoru Omichi, Koichi Fuk ...1996 年 120 巻 5 号 p. 1002-1006
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xylα1-3Gle) and a trisaceharide (Xylα1-3Xylα1-3Gle) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors. The structures of these sugar chains suggested the presence of an α1→3xylosyltransferase for their biosynthesis. We report here on the detection of α1→3xylosyltransferase activity which catalyzes the transfer of xylose to Xylαl-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xylal-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor. The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xylα1-3Xylα1-3Glc by Smith degradation, mass spectrometry, and α- and β-xylosidase digestions. The apparent Km value for pyridylaminated Xylα1-3Glc was 52mM and for UDP-D-Xyl 0.28mM. Optimum pH was 7.2. The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xylα1-3Glc, forming Xylα1-3Xylα1-3Glc in human cells.抄録全体を表示PDF形式でダウンロード (518K) -
Hafiz Ahmed, Nilda E. Fink, Jan Pohl, Gerardo R. Vasta1996 年 120 巻 5 号 p. 1007-1019
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーSelected biochemical properties, including the charge heterodispersity profile and carbohydrate specificity, of bovine galectin-1 were determined in detail. The lectin was purified through an improved purification protocol that yielded 35-40mg/kg of wet tissue with a specific activity of 1.7-2×104mg-1•ml. The galectin is a homodimer of approximately 14.5 kDa subunits with Emg/ml280 of 0.65ml•mg-1•cm-1. When stored in the presence of its carbohydrate ligand, the lectin's binding activity remained stable in a non-reducing environment even at room temperature. The optimal pH for binding to the ligand was 6.5-8.0. The overall carbohydrate specificity of the bovine galectin-1 isolated from spleen is similar to that of the galectin isolated from heart and to other mammalian galectins that exhibit “conserved” (Type I) carbohydrate recognition domains (CRDs) [Ahmed, H. and Vasta, G. R. (1994) Glycobiology 4, 545-549], but differs from those from Xenopus laevis and rat intestine domain I. The fluorescence of 4-methylumbelliferyl α-D-galactopyranoside was quenched on binding to bovine spleen galectin-1. Scatchard plots of data obtained at 5, 15, and 30°C showed that the galectin has two sugar exothermic binding sites with association constants of 3.4×105, 1.0×105, and 0.3×105, respectively. Chemical modification studies indicated that histidine, tryptophan, carboxylic acid, and arginine, but not lysine or tyrosine, are involved in the binding to the carbohydrate ligand. On isoelectric focusing, the spleen galectin-1 appeared as six isoforms ranging from pI 4.56-4.88 with main components at pI 4.63 (34.0%), 4.73 (42.6%), and 4.88 (16.6%). The galectin-1 isolated from heart yielded a quali- and quantitatively different profile with four isoforms ranging from pI 4.53-4.73, those with pls of 4.56, 4.63, and 4.73 being common to the spleen homolog. Edman degradation of selected peptides purified from the spleen galectin-1 digest revealed amino acid sequences identical to those obtained for the heart galectin-1. This suggests that although point mutations in the subunit primary structure may not be the likely source of isolectins, as observed for X. laevis, tissue-specific co- or post-translational modifications may be the possible cause of the differences in the galectin isoform profile between bovine spleen and heart.抄録全体を表示PDF形式でダウンロード (2586K) -
Yumiko Watanabe, Kiyomitsu Nara, Hiroshi Takahashi, Yoshitaka Nagai, Y ...1996 年 120 巻 5 号 p. 1020-1027
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe have cloned the cDNA for a GD3 synthase (α-2, 8-sialyltransferase, [EC 2. 4. 99. 8]) from a rat embryonic brain cDNA library. Mammalian cells transfected with the cloned cDNA expressed GD3 on the cell surface and showed GD3 synthase activity. The deduced protein (342 amino acid residues) was predicted to have a type II membrane topology containing the “sialyl motif” and was found to be 91% similar to its human homologue. Analysis of the acceptor specificity of GD3 synthase protein indicated that this enzyme catalyzed the biosynthesis of GT1a and GQ1b as well as GD3. Northern blot analyses showed that the GD3 synthase gene is preferentially transcribed in the brain and the spleen. The expression of GD3 synthase mRNA was developmentally regulated, with the highest level in the brain during embryonic days 15 to 18. In situ hybridization analyses demonstrated that the GD3 synthase is strongly expressed in the ventricular/subventricular zone of the embryonic rat brain and retina. In the adult rat, GD3 synthase mRNA was detected in the cerebral cortex, hippocampus, thalamus, and cerebellum. These studies show that the spatio- and stagerestricted expression of GD3 in the developing rat brain may be regulated in part by the level of GD3 synthase mRNA.抄録全体を表示PDF形式でダウンロード (4224K) -
Eiji Sakashita, Hiroshi Sakamoto1996 年 120 巻 5 号 p. 1028-1033
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe Drosophila Sex-lethal (Sxl) contains two RNA-binding domains (RBDs) which belong to the RNA recognition motif (RRM) group. Sxl binds to a specific uridine-rich sequence which is believed to be the major cis-acting element for the splicing regulation of the transformer (tra) mRNA precursor. Here we show evidence supporting the previous suggestion that Sxl recognizes the sequence context downstream of the uridine-rich sequence. In addition, by means of UV-crosslinking assays with Sxl deletion constructs, we have demonstrated that Sxl RNA binding requires both of its RBDs for specificity and strength. Moreover, by the yeast two-hybrid analysis, we found that homodimeric interaction occurs between two Sxl molecules. Interestingly, the amino- and carboxy-terminal regions outside of the Sxl RBDs are dispensable for such dimerization, indicating that the protein-protein interaction is also mediated by RBDs. Coprecipitation experiments in vitro showed that the protein-protein interaction seems to be RNA-dependent but greatly enhanced by addition of the specific RNA containing the Sxl binding site, suggesting that the conformational change which is induced on binding to RNA may facilitate the interaction between Sxl molecules.抄録全体を表示PDF形式でダウンロード (1909K) -
Jun-ichiro Tsutsui, Maiko Moriyama, Naomichi Arima, Hideo Ohtsubo, Hir ...1996 年 120 巻 5 号 p. 1034-1039
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーCadherins are Ca2+-dependent cell-cell adhesion molecules, and are involved in the formation and maintenance of the histo-architecture. Using cultured human leukemia cell lines (adult T cell leukemia and thymus-derived lymphoma cell lines), we obtained evidence that cadherins and catenins are expressed in these cell lines but not in normal leukocytes. Immunoblot analysis of cells using a pan-cadherin serum, directed against the conserved carboxyl-terminus of cadherins, revealed a major band of 130 kDa and a minor one of 135 kDa. The 130 kDa cadherin was also recognized by anti-N-cadherin antibodies. A human N-cadherin cDNA probe hybridized to a 4.3 kb mRNA isolated from cells immunologically positive for N-cadherin. Sequencing of the cDNA fragments isolated from the cells revealed a N-cadherin sequence. Cell surface expression of N-cadherin was confirmed by indirect immunofluorescence staining of the cells. Immunoblot and Northern blot analyses also revealed the presence of α-catenin, β-catenin, and γ-catenin (plakoglobin) in these cell lines. Immunoprecipitation with anti-N-cadherin antibodies and subsequent immunoblot analysis with anti-catenin antibodies revealed that N-cadherin is associated with α- and β-catenins, a prerequisite for cadherins to be functional. These results suggest an important role of the cadherin-catenin complexes in the behavior of the leukemia cells.抄録全体を表示PDF形式でダウンロード (3196K) -
Rho Min-Seok, Yuji Kawamata, Hidemitsu Nakamura, Akinori Ohta, Masamic ...1996 年 120 巻 5 号 p. 1040-1047
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーSaccharomyces cerevisiae mutants that were unable to utilize extracellular ethanolamine for phosphatidylethanolamine synthesis were isolated. Two of them carried recessive chromosomal mutations in a same gene and were defective in CTP:phosphoethanolamine cytidylyltransferase (ECT) activity in vitro (Ect-). In an Ect- mutant that also carried the cho1 mutation, phosphatidylethanolamine accounted for less than 2% of total phospholipids, suggesting the importance of ECT in phosphatidylethanolamine synthesis. By screening a genomic library on a low copy number vector, three complementary clones of different size were isolated. A 2.8-kb common DNA region carried an open reading frame (ORF) of 969 by in length, of which a truncated form failed to complement the Ect- mutation. This ORF was identical to the previously isolated MUQ1 gene of unknown function. Its deduced amino acid sequence had significant similarity to CTP:phosphocholine cytidylyltransferases of yeast and rat. The entire ORF, when combined with the glutathione Stransferase gene and expressed in Escherichia coli, exhibited ECT activity. These results indicate that the cloned gene encodes a catalytic subunit of ECT of S. cerevisiae.抄録全体を表示PDF形式でダウンロード (3859K) -
Hiroyuki Suzuki, Kazuhiro Takahashi, Ken-ichi Yasumoto, Nobuo Fuse, Sh ...1996 年 120 巻 5 号 p. 1048-1054
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe have cloned the full-length cDNA encoding the rat homolog of type I neurofibromin isoform, a protein product of a gene linked to neurofibromatosis type 1. Rat type I neurofibromin isoform is composed of 2, 820 amino acid residues and shares about 98.5% amino acid identity with the human counterpart. By S1 nuclease mapping analysis of the alternatively spliced neurofibromin mRNAs in adult rat tissues, we showed that type I isoform mRNA was predominantly expressed in the brain, pituitary, and testis, while type II mRNA, carrying a 63-nucleotide insertion in the region coding for the domain related to GTPase-activating protein, was predominantly expressed in most other tissues, such as heart, kidney, and ovary. Furthermore, type II mRNA is predominant in the testis at age 1 week, while type I mRNA becomes predominant at 3 weeks and is subsequently expressed at higher levels, as seen in the adult testis. In contrast, type I mRNA is the predominant form in the brain throughout the postnatal development. Thus, the relative expression levels of type I and type II mRNAs may be specific to the tissues and to the developmental stage of certain tissues.抄録全体を表示PDF形式でダウンロード (3414K)
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