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

Na， k - atp酶由α亚基和β亚基组成，其化学计量比为1比1。α -亚基包含了瓦巴因的结合位点和催化位点，而β -亚基的功能尚未完全解决。由于酶只分布在基侧质膜上，因此可以假设β -亚基在酶的极化定位中起作用。近年来研究表明，胃H， k - atp酶由α亚基和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作用似乎与Na， k - atp酶的亚基相似。在这些发现的基础上，我们试图在接下来的实验中阐明β -亚基对这些atp酶的作用。(1)在神经生长因子（NGF）的作用下培养PC12细胞，细胞内Na、k - atp酶活性在培养第6天提高了2倍。通过免疫印迹分析，NFG存在下的PC12细胞α亚基和β亚基的相对含量分别比ngf处理前的细胞含量增加了2.5倍（α亚基）和5.5倍（β亚基）。脉冲标记实验表明，ngf处理前细胞新合成的β与α的摩尔比为0.5，处理后为1。这些结果表明PC12细胞中的β亚基可能在NGF诱导Na， k - atp酶的过程中起重要作用。(2)应用免疫电镜定量观察了Na、k - atp酶在大鼠眶外泪腺、前庭器官和肾脏中的定位。(3)根据所报道的氨基酸序列合成大鼠胃H， k - atp酶-亚基肽和结肠H， k - atp酶-亚基肽，注射家兔，ELISA分析所得抗血清效价高。少

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