イオン・溶質輸送と共役ネットワーク

1994 Fiscal Year Annual Research Report

• Project/Area Number: 04266101
• Research Institution: Tokyo Metropolitan Organization for Medical Research
• Principal Investigator: 川喜田 正夫 財団法人東京都臨床医学総合研究所, 医化学研究部門, 研究員 (00012740)
• Co-Investigator(Kenkyū-buntansha): 原 諭吉 東京医科歯科大学, 医学部, 助教授 (00092429) ; 山口 明人 千葉大学, 薬学部, 助教授 (60114336) ; 重川 宗一 国立循環器センター研究所, 循環分子生理部, 研究員 (00113738) ; 青島 均 山口大学, 教養部, 教授 (20089907) ; 乾 賢一 京都大学, 医学部, 教授 (70034030)
• Keywords: 共役物質輸送 / Na^+ / H^+アンチポーター / H^+ / ジペプチドシンポーター / Band 3 / テトラサイクリン / H^+アンチポーター / アラニンキャリアー / バクテリオロドプシン / Na^+ / K^+-ATPase

Research Abstract

細胞の膜系における各種のイオンや溶質の共役輸送系の実体とそのネットワークに関して、以下の結果を得た。
1.Na^+/H^+-アンチポーター(NHE)のisoformに対する特異的ペプチド抗体を作成し、各isoformの組織における局在性を明らかにした(川喜田)。NHE1 isoformの活性の細胞内pHによる調節の情報がカルモジュリンによって伝達されることを示し、調節に関与する領域を同定した(重川)。腸炎ビブリオNHEのアミロライド結合部位の変異体では、NHEのイオン特異性が同時に変化することを明らかにした(土屋)。
2.H^+/ジペプチドシンポーターのcDNAクローニングに成功し、塩基配列を決定した(乾)。腎上皮細胞による有機アニオンの経細胞輸送のin vitro実験系を確立し、プロテインキナーゼCによる活性調節を明らかにした(高野)。
3.赤血球膜のアニオン輸送体Band 3の構造をタンパク質化学的に解析し、新しい構造モデルを提案した。(濱崎)。
4.テトラサイクリン/H^+アンチポーターのトポロジーおよび反応機構を、復帰変異解析、Cys走査変異体の解析によって詳しく研究し、精細な構造モデルを発表した(山口)。
5.好熱菌のアラニンキャリアーの大量発現に成功した。さらに結晶化による詳細な構造解析の方向を指向している(平田)。
6.Na^+/K^+-ATPaseのαβ分子集合体形成機構について、H^+/K^+-ATPaseとのキメラを用いて解析し、N末50残基については互換性があることを明らかにした(川村)。また、α1およびα3 isoformの間の特性のちがいがNa^+遊離サイトの基質親和性のちがいに基づくものであることを明らかにした(原)。
7.バクテリオロドプシンのH^+移動反応に関与するアミノ酸残基として、Asp85,Asp212,Asp96/Thr46,Trp182を同定することに成功した(前田)。

Research Products

• [Publications] Ono,A: "Transport of envelope proteins of Sendai virus HN and FO,is blocked at different steps by thapdigargin and other perturbants to intracellular Ca2+" J.Biochem.116. 649-656 (1994)
• [Publications] Aoki,K: "Isolation and characterization of a novel mutant mouse cell line resistant of Newcastle disease virus:constitutive interferon production and enhanced interferon sensitivity" Arch.Virol.139. 337-350 (1994)
• [Publications] Suzuki,T: "Sites of labeling with N-(3-pyrene)maleimide on Ca^<2+>-transporting ATPase of the Saroplasmic reticulum" J.Biochem.117. 881-887 (1995)
• [Publications] Wada,M.: "Molecular cloning of P-type ATPases on intracellular membranes of the marine alga Heterosigma akashiwo" Plant Mol.Biol. 26. 699-708 (1994)
• [Publications] Sumimoto,H.: "Role of Src homology domains in assembly and activation of the phagocyte NADPH oxidase" Proc.Natl.Acad.Sci.,U.S.A.91. 5345-5349 (1994)
• [Publications] Hashimoto,Y.: "Functional evidence for an apical ANP receptor in LLC-PK_1 kidney epithelial cells" Eur.J.Pharmacol. 268. 443-445 (1994)
• [Publications] Matsumoto,S.: "Transcellual transport of oral cephalosporins in human intestinal epithelial cells,Caco-2:interaction with dipeptide transport systems in apical and basolateral membranes" J.Pharmacol.Exp.Ther.270. 498-504 (1994)
• [Publications] Sasaki,S.: "Cloning,characterization,and chromosomal mapping of human aquaporin of collecting duct" J.Clin.Invest.93. 1250-1256 (1994)
• [Publications] Aoshima,H.: "Effects of aliphatic alcohols and food additives on nicotinic acetylcholine receptors in Zenopus oocytes" Biosci.Biothech.biochem.58. 1776-1779 (1994)
• [Publications] Okubo,K.: "Red blood cell Band 3" J.Biol.Chem.269. 1918-1926 (1994)
• [Publications] Kato,S.: "Analysis of mdr-1 gene expression in human leukemic cells by quantitative PCR" Leukemia and Lymphoma. 14. 129-135 (1994)
• [Publications] Ideguchi,H.: "A comprehensive scheme for the systematic investigation of hemolytic anemia" Ann.Clin.Lab.Sci.24. 412-421 (1994)
• [Publications] M.Takano: "Transport of gentamicin and fluid-phase endocytosis markers in the LLC-PK_1 kidney epithelial cell line" J.Pharmacol.Exp.Ther.268. 669-674 (1994)
• [Publications] M.Takano: "Bestatin transport in rabbit intestinal brush-border membrane vesicles" Biochem.Pharmacol.47. 1089-1090 (1994)
• [Publications] M.Takano: "Cellular toxicity of aminoglycoside antibiotics in G418-sensitive and -resistant LLC-PK_1 cells" Pharm.Res.11. 609-615 (1994)
• [Publications] M.Takano: "p-Aminohippurate transport in apical and basolateral membranes of the OK kidney epithelial cells" J.Pharmacol.Exp.Ther.269. 970-975 (1994)
• [Publications] M.Takano: "Differential sensitivity of organic anion transporters in rat renal brush-border membrane to diethyl pyrocarbonate" Biochim.Biophys.Acta. 1192. 190-196 (1994)
• [Publications] Y.Tomita: "Transport of oral cephalosporins by the H^+/dipeptide cotransporter and distribution of the transport activity in isolated rabbit intestinal epithelial cells." J.Pharmacol.Exp.Ther.272. 63-69 (1995)
• [Publications] Wakabayashi,S.: "Growth factor activation and "H^+-sensing" of the Na^+/H^+ exchange isoform 1(NHE1).Evidence for an additional mechanism not requiring direct phosphorylation." J.Biol.Chem.269. 5583-5588 (1994)
• [Publications] Bertrand,B.: "The Na^+/H^+ exchanger isoform 1(NHE1)is a novel member of calmodulin binding proteins." J.Biol.Chem.269. 13703-13709 (1994)
• [Publications] Wakabayashi,S.: "Mutation of calmodulin-binding site renders the Na^+/H^+ exchanger(NHE1)highly H^+-sensitive and Ca^<2+> regulation-defective." J.Biol.Chem.269. 13710-13715 (1994)
• [Publications] Ohsima,N.: "Regulation of Ca^<2+> entry in rat aortic smooth muscle cells in primary culture." J.Biochem.116. 274-281 (1994)
• [Publications] Iwata,Y.: "Ca^<2+>-ATPase distributes differently in cardiac sarcolemma than dihydropyridine receptor α 1 subunit and Na^+/Ca^<2+> exchanger." FEBS Lett.355. 65-68 (1994)
• [Publications] 岩本隆宏: "培養血管平滑筋細胞L型電位依存性Ca^<2+>チャンネルのリン酸化による制御" 血管. 17. 93-101 (1994)
• [Publications] I.Yamato: "Site-specific alteration of arginine-376,the unique positively charged amino acid residue in the mid-membrane spanning regions of the proline carrier of E.coli." J.Biol.Chem.269. 5720-5724 (1994)
• [Publications] K.Takase: "Sequencing and characterization of the ntp gene cluster for vacuolar type Na^+-translocating ATPase of Enterococcus hirae." J.Biol.Chem.269. 11037-11044 (1994)
• [Publications] 山登一郎: "タンパク質をデザインする一生命科学における第2の暗号解読問題" Sut Bulletin. 8. 36-41 (1994)
• [Publications] Y.Komeiji: "Clycine-85 of the trp-repressor of E.coli is important in forming the hydrophobic tryptophan binding pocket:experimental and computational approaches." Prot.Engng.7. 1239-1247 (1994)
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• [Publications] 丸山明子: "酸化LDLのスーパーオキサイド産生能及び好中球からのスーパーオキサイド産生誘導能" 帝京医学雑誌. 17. 511-521 (1994)
• [Publications] Noriko Okazaki: "Characterizaion of the lactose transport system in Citrobacter freundii." Biol.Pharm.Bull.17(6). 794-797 (1994)
• [Publications] Teruo Kuroda: "Properties of the Na^+/H^+ antiporter in Vibrio parahaemolytics." J.Biochem.115. 1162-1165 (1994)
• [Publications] Toshi Shimamoto: "The NhaB Na^+/H^+ antiporter is essential for intracellular pH regulation under alkaline condition in Escherichia coil." J.Biochem.116. 285-290 (1994)
• [Publications] Teruo Kuroda: "Properties and sequence of the NhaA Na^+/H^+ antiporter of Vibrio parahaemolyticus." J.Biochem.116. 1030-1038 (1994)
• [Publications] Jong-ln Lee: "C loning and sequence of the gene for the lactose carrier of Citrobacter freundii." Biochem.Biophys.Res.Commun.203(3). 1882-1888 (1994)
• [Publications] Rafiquel l.Sarker: "Characterization of a glucose transport system in Vibrio parahaemolyticus." J.Bacteriol.176. 7378-7382 (1994)
• [Publications] Kamata,H.: "Amphiphilic Peptides Enhance thd Efficiency of Liposome-mediated DNA Transfection." Nucleic Acids Research. 22. 536-537 (1994)
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• [Publications] Yagisawa.H.: "Expression and Characterization of an Inositol 1,4,5-Trisphosphate Binding Domain of Phosphatidylinositol-specific Phospholipase C-δ 1" J.Biol.Chem.269. 20179-20188 (1994)
• [Publications] Shinomiya,H.: "Identification of the 65-kDa Phosphoprotein in Murine Macrophages As a Novel Protein:Homology with Human L-Plastin." Biochem.Biophys.Res.Commun.202. 1631-1638 (1994)
• [Publications] Ohta,T.: "Molecular Cloning of Two New Heat Shock Genes Related to the hsp70 Genes in Staphylococcus aureus." J.Bacteriol.176. 4779-4883 (1994)
• [Publications] Maeda,A.: "Interaction of aspartate-85 with a water molecule and the protonated Schiff base in the L intermediate of bacteriorhodopsin:A Fourier transform infrared spectroscopic study." Biochemistry. 33. 1713-1717 (1994)
• [Publications] Sasaki,J.: "Complete identification of C=O stretching vibrational bands of protonated aspartic acid residues in the difference infrared spectra of M and N intermediates versus bacteriorhodpsin." Biochemistry. 33. 3178-3184 (1994)
• [Publications] Brown,L.S.: "The proton transfers in the cytoplasmic domain of bacteriorhodopsin are facilitated by a cluster of interacting residues." J.Mol.Biol.239. 401-414 (1994)
• [Publications] Brown,L.S.: "The retinal Schiff base-counterion complex of bacteriorhodopsin:Changed geometry during the photocycle is a cause of proton transfer to aspartate 85." Biochemistry. 33. 12001-12011 (1994)
• [Publications] Kataoka,M.: "Energy coupling in an ion pump:the reprotonation switch of bacteriorhodopsin." J.Mol Biol.243. 621-638 (1994)
• [Publications] Yamazaki,Y.: "Interaction of tryptophan-182 with the retinal 9-methyl group in the L intermediate of bacteriorhodopsin." Biochemistry. 34. 577-582 (1995)
• [Publications] Yamaguchi,A.: "Hot spots for sulfhydryl inactivation of Cys mutants in the widely conserved sequence motifs of the meta-tetracycline/H^+....." J.Biochem.115. 958-964 (1994)
• [Publications] Sato,K.: "Tetracycline/H^+ antiporter was degraded rapidly in Escherichia coli cells when truncated at last transmembrane helix ....." Biochem.Biophys.Res.Commun.202. 258-264 (1994)
• [Publications] Someya,Y.: "Site-specificity of the second-site suppressor mutation of the Asp285- Asn mutant of metal-tetracycline/H^+ antiporter of ....." Biochemistry. 34. 7-12 (1995)
• [Publications] Someya,Y.: "A novel glycylcycline,9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline,is neither transported nor recognized by.." Antimicrob.Agents Chemother.39. 247-249 (1995)
• [Publications] Kawano,H.: "Phenytoin,antiepileptic drug,competitively blocked non-NMDA receptors produced by Xenopus oocytes." Neuroscience Lett.166. 183-186 (1994)
• [Publications] Noguchi,S.: "The functional roles of disulfide bonds in the β-subunit of (Na,K)ATPase as studied by site-directed mutagenesis" FEBS Lett.341. 233-238 (1994)
• [Publications] Morohashi,M.: "Expression of (Na,K)ATPase/Ca-ATPase chimeric proteins in Xenopus oocytes revealed that the COOH-terminal one-third of the (Na,K)ATPase α-subunit is involved in assembly with the β-subunit" J.UOEH. 16. 277-286 (1994)
• [Publications] Ueno,S.: "Functional consequences of substitution of the disulfide-bonded segment,cys127cys150,located in the extracellular domain of the Na,K-ATPase β subunit:arg148 is essential for the functional expression of Na,K-ATPase" J.Biochem. (in press). (1995)
• [Publications] 濱崎直孝: "赤血球膜バンド3蛋白質の構造と機能に関する研究" 福岡医学雑誌. 85. 335-340 (1994)
• [Publications] Ohkita,Y.J.: "Changes in structure of the chromophore in the photochemical process of bovine rhodopsin as revealed by FTIR spectroscopy for hydrogen-out-of-plane vibrations." Biophys.Chem.(in press). (1995)
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• [Publications] Sasaki,J.: "Nanosecond time-resolved infrared spectroscopy distinguishes two K species in the bacteriorhodopsin photocycle." Biophys.J.(in press). (1995)
• [Publications] Hara,Y.: "The Sodium Pump" Molecular cloning of P-type ATPases on intracellular membranes of the marine alga Heterosigma akashiwo, 906 (1994)
• [Publications] 乾賢一: "膜学実験シリーズ第1巻 生体膜編、日本膜学会編" 共立出版, 424 (1994)
• [Publications] 山登一郎: "薬学研究の進歩10巻" 薬学研究奨励財団, (1994)
• [Publications] A.Suenaga: "Progress in Cell Research:Molecular Biophysics of Ion Channels(in press)" Elsevier Sci.Pub., (1995)