| Co-Investigator(Kenkyū-buntansha) |
TANIGUCHI Kazuya Hokkaido University School of Dentistry, 歯学部, 助教授 (40028204)
HARA Yukichi Tokyo Medical and Dental University School of Medicine, 医学部, 助教授 (00092429)
NAKAO Makoto Tokyo Medical and Dental University School of Medicine, 医学部, 教授 (10013781)
KAWAMURA Masaru University of Occupational and Environmental Health, 教授 (00049066)
NAGANO Kei Jichi Medical School, 教授 (70048940)
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| Budget Amount *help |
¥11,000,000 (Direct Cost: ¥11,000,000)
Fiscal Year 1987: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1986: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Research Abstract |
1.Molecular Structure : Primary structure of and chains from torpedo and human Na^+,K^+-ATPase were deduced from the cloned cDNA's. Structures of three isoforms, , (+) and III were also deduced in rat brain. mRNA's of and chains synthesized from the cDNA's were injected into Xenopus oocytes, and the translated products could exhibit the Na^+,K^+-ATPase activity and the Na pump activity. Although each translated and chain independently had an incorporation ability to the membrane, co-existence of with was necessary for the expression of the physiological function with Na^+,K^+-ATPase by lowangle laser light scattering photometry coupled with HPLC, the change of protomer interaction was proved to corelate with the conformational change of the enzyme during turnover.
2.Enzyme Reaction Mechanism : Extrinsic fluorescence of BIPM, FITC, IAF and DACM, intrinsic fluorescence of Trp, light scattering and ^<42>K^+ binding were measured in each of the reaction intermediates, and the conformational
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change of the enzyme was analyzed with the above parameters. Inhibition by amine compounds was investigated to know the nature of cation sites. 3.Transport Function : H^+ transport could be substituded in Na^+ transport by Na pump with the reconstructed vesicles. Insulin increased Na^+ eflux with frog skeletal muscle, and the increase was concluded to be due to the increase in the number of Na^+,K^+-ATPase molecule which was translocated to the plasma membrane from the inside of cell. During maturation of the primary cultured neurons from rat, (+) enzyme was induced, and the (+) pump activity functioned preferentially than the pump activity. mRNA's for , (+) and III were determined with cDNA probes in rat brain, heart and kidney, and the changes in the content were examined in the course of cell culture. Presence of three kind of cation co-transport systems, i.e., Na^+-dependent, H^+ dependent, and Na^+ or H^+-dependent system, was demonstrated in the brush border membrane of intestin. Less
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