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)
|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1992 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1991 : ¥1,100,000 (Direct Cost : ¥1,100,000)
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