Studies on an anion-translocating protein in a biological membrane - Comparison of a Cl -translocating ATPase with other cation-translocating ATPases

1.（1）生化研究：建立了髋臼菌蛋白质SDS-PAGE电泳后的高效电洗脱方法。从Cl^--ATPase的a亚基（54 kDa）和B亚基（50 kDa）获得的部分氨基酸序列与高等植物叶绿体ATPase的a亚基和b亚基分别具有40 - 100%的相似性。Cl^--ATP酶在脂质体中的重组得到改善，这表明Cl^--的转运需要2个Cl^-与1个酶的结合，而Br^-和F^-则减弱了由ATP驱动的Cl^--转运活性。(2)分子克隆：一个推定的片段（约。通过直接扩增髋臼cDNA文库，获得了编码Cl^--ATP酶α亚基的cDNA片段（约160 bp）。推导的氨基酸序列从N端开始与蛋白质序列完全一致。还获得了编码B亚基的几个候选片段，目前正在进行DNA测序。 ...更多信息 2.髋臼中的其他阳离子转运ATP酶叶绿体ATP酶，α-γ-复合体的纯化和性质。其性质与Cl^--ATPase不同。通过H^+-转运活性和免疫反应性鉴定了绿豆液泡ATP酶和无机焦磷酸酶。对编码CF_1-ATP酶α亚基（270 bp）和β亚基（270 bp）以及液泡膜ATP酶大亚基（280 bp）和小亚基（690 bp）的PCR产物进行了鉴定，推导的氨基酸序列与高等植物中相应亚基的同源性最高（90%以上）.髋臼硫酸盐渗透酶人工海水中硫酸盐剥夺24 h后，硫酸盐渗透酶活性下降.约3倍的增长，在<35>4SO_4 ~+<2->吸收由成年细胞。PCR扩增得到一个359 bp的片段，与构巢拟囊藻硫酸盐通透酶复合物中的CysA蛋白同源性为55%。少

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. … More 2. Other cation-translocating ATPases in Acetabularia acetabulumChloroplast 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 acetabulumSulfate 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