Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||Faculty of Pharmaceutical Sciences, Okayama University|
OHMORI Shinji Fac. Pharm. Sci., Okayama Univ., Professor, 薬学部, 教授 (10032872)
SHIOZAWA Jud マックス, プランク生化学研究所・膜生化学部門, 研究員
LOTTSPEICH Friedrich Max-Planck-Inst. of Biochemistry, Prof., プランク生化学研究所・遺伝子センター蛋白質化学部門, 教授
OESTERHELT Dieter Max-Planck-Inst. of Biochemistry, Director (Prof.), プランク生化学研究所・膜生化学部門, 教授
MAEDA Masatomo Inst. of Scientific and Industrial Res., Osaka Univ., Assoc. Prof., 産業科学研究所, 助教授 (80190297)
FUTAI Masamitsu Inst. of Scientific and Industrial Res., Osaka Univ., Prof., 産業科学研究所, 教授 (50012646)
IKEDA Mikiko Fac. Pharm. Sci, . Okayama Univ., Assoc. Prof., 薬学部, 助教授 (20112154)
SHIOZAWA Judith A. Max-Planck-Inst. of Biochemistry, Res. Assoc.
|Project Period (FY)
1990 – 1991
Completed(Fiscal Year 1991)
|Budget Amount *help
¥7,800,000 (Direct Cost : ¥7,800,000)
Fiscal Year 1991 : ¥4,000,000 (Direct Cost : ¥4,000,000)
Fiscal Year 1990 : ¥3,800,000 (Direct Cost : ¥3,800,000)
|Keywords||Acetabularia / Cl^--ATPase / Chloroplast ATPase / Vacuolar ATPase / Pyrophosphatase / Sulfate permease / Molecular cloning / Cl^-輸送性ATPase / 液胞膜ATPase / 遣伝子クロ-ニング / 生化学的解析 / 蛋白質化学的解析|
1. Cl^- -translocating ATPase in Acetabularia acetabulum
(1) Biochemical studies : Preparative high-yield electroelution method of proteins after SDS-PAGE was established. Partial amino acid sequences obtained from the a (54 kDa) and b (50 kDa) subunit of the Cl^--ATPase showed 40 to 100% similarity to the and subunit of chloroplast ATPase in higher plants, respectively. The improved reconstitution of the Cl^--ATPase into liposomes revealed that the binding of 2 Cl^- to 1 enzyme was required for the Cl^--transport, and Br^- and F^- diminished the Cl^--transport activity driven by ATP.
(2) Molecular cloning : A putative fragment (ca. 160 bp) encoding the a subunit of the Cl^--ATPase was identified by the use of direct amplification of cDNA library from Acetabularia acetabulum. The deduced amino acid sequence contained the identical sequence with the protein sequence from the N-terminus. Several candidate-fragments encoding the b subunit were also obtained and are now under DNA sequencing.
2. Other cation-translocating ATPases in Acetabularia acetabulum
Chloroplast ATPase, alphabetagamma-complex was purified and characterized. The characteristics were different from the Cl^--ATPase. A vacuolar ATPase and inorganic pyrophosphatase were identified by the H^+-transport activities and immunoreactivities with the antibodies against the large and small subunits of mung bean vacuolar ATPase, and pyrophosphatase. The PCR products encoding the alpha (270 bp) and beta (270 bp) subunits of CF_1-ATPase, and the large (280 bp) and small (690 bp) subunits of vacuolar ATPase were identified, and the deduced amino acid sequences showed the highest similarity (over 90%) to the respective subunit of higher plants.
3. Sulfate permease in Acetabularia acetabulum
Sulfate deprivation for 24 h from the artificial sea water caused. about 3-fold increase in ^<35>so_4^<2-> uptake by grown-up cells. A 359 bp fragment amplified by PCR method showed 55% identities to Anacystis nidulans CysA protein-involved in sulfate permease complex. Less