TASHIRO Yutaka Kansai Med.Univ., Dpt.of Physiol., Professor, 医学部, 教授 (40077558)
YOSHIMORI Tamotsu Kansai Med.Univ., Dpt.of Physiol., Assistant, 医学部, 助手 (60191649)
YAMAMOTO Akitsugu Kansai Med.Univ., Dpt.of Physiol., Lecturer, 医学部, 講師 (30174775)
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
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