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Functional analysis of the beta-subunits of Na, K-ATPase and H, ATPase

Na, K-ATPase 和 H, ATPase β 亚基的功能分析

基本信息

批准号：
03833032
负责人：
OMORI Koichiro
金额：
$ 1.09万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03833032/
关键词：
Na, K-ATPase H, ATP-ase PC12 Cell Nerve Growth Factor Vestibular Apparatus Exorbital Lacrimal Gland Kidney 腎臓 Na,KーATPase A23187 nerve growth factor

项目摘要

Na,K-ATPase consists of the alpha- and beta-subunits with the stoichiometry of one to one. alpha-subunit contains the binding site for ouabain as well as the catalytic site, whereas the function of the beta-subunit is not yet completely solved. As the enzyme exclusively distributes on the basolateral plasma membrane, it is possible to suppose that the beta-subunit plays a role for the polarized localization of the enzyme. Recently, it has been evident that gastric H, K-ATPase is comprised of the alpha- and beta-subunits and very close to NA, K-ATPase in structure. Therefore, the functional roles of both the subunits of H, K-ATPase are seemed to be similar to those of Na, K-ATPase subunits. On the bases of these findings, we tried to elucidate the functions of the beta-subunit on these ATPases in the following experiments.(1) When PC12 cells were cultivated in the presence of nerve growth factor (NGF) which is known to induce a neuron-like morphological and physiological differentiation … More of the cells, Na, K-ATPase activities in those cells increased by -2 times on day 6 in the culture. The relative contents of the alpha- and beta-subunits of PC12 cells in the presence of NFG, when analyzed by immunoblot, were increased by 2.5-fold (alpha-subunit) and 5.5-fold (beta-subunit) over the contents of cells berfore NGF-treatment, respectively. Pulse labeling experiments showed that the molar ratio of beta to alpha newly synthesized was 0.5 in the cells before NGF-treatment and was 1 after the treatment. These results indicate that the beta-subunit in PC12 cells may play an important role in the induction of Na, K-ATPase by NGF.(2) Localization of Na, K-ATPase in rat exorbital lacrimal gland, vestibular apparatus and kidney were investigated quantitatively by immuno-electron microscopy.(3) Synthetic peptides of rat gastric H, K-ATPase beta-subunit and colon H, K-ATPase alpha-subunit were made according to the reported amino acid sequences these peptides were injected into rabbits and the obtained anti-serum showed high titer by ELISA analyses. Less

Na，K-ATP酶由化学计量比为1：1的α亚基和β亚基组成。α-亚基含有哇巴因的结合位点以及催化位点，而β-亚基的功能尚未完全解决。由于该酶仅分布在基底外侧质膜上，因此可以假设β亚基对该酶的极化定位起作用。近年来研究表明，胃H，K-ATP酶由α-和β-亚基组成，其结构与NA，K-ATP酶非常接近。因此，H，K-ATP酶的两个亚基的功能与Na，K-ATP酶的功能相似。基于这些发现，我们在以下实验中试图阐明这些ATP酶的β亚基的功能。(1)当在已知诱导神经元样形态和生理分化的神经生长因子（NGF）存在下培养PC 12细胞时， ...更多信息在培养的第6天，这些细胞的Na，K-ATP酶活性增加了约2倍。当通过免疫印迹分析时，在NFG存在下的PC 12细胞的α-和β-亚基的相对含量分别增加了2.5倍（α-亚基）和5.5倍（β-亚基），超过了在NGF处理之前的细胞的含量。脉冲标记实验表明，新合成的β与α的摩尔比在神经生长因子治疗前的细胞中为0.5，治疗后为1。结果提示，PC_（12）细胞β亚基在NGF诱导Na，K-ATP酶活性中可能起重要作用。(2)应用免疫电镜技术对大鼠眶外泪腺、前庭器和肾脏内Na，K-ATP酶的定位进行了定量研究。(3)根据已报道的氨基酸序列，合成了大鼠胃H，K-ATP酶β亚基和结肠H，K-ATP酶α亚基的多肽，并将其注射家兔，获得了高效价的抗血清。少