| Project/Area Number |
03454543
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | Hokkaido University |
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Principal Investigator |
TANIGUCHI Kazuya Hokkaido Univ., faculty of Science, Prof., 理学部, 教授 (40028204)
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| Project Period (FY) |
1991 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1993: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1992: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1991: ¥3,500,000 (Direct Cost: ¥3,500,000)
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| Keywords | Na^+, K^+ATPase / Na^+, K^+-ATPase / Conformation change / Sodium pump / Ion trnsport pump / Proton pump / 蛍光プローブ / エネルギー移動 / 構造変化 / ATP加水分解酵素 |
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| Research Abstract |
A preparation of pig kindney Na^+, K^+-ATPase showed changes in the fluorescence energy transfer between fluorescent probes in the alpha-subunits. Excitation (305 nm) of the N-(p-(2-benzimidazolyl)phenyl) maleimide(BIPM) probe at Cys-964, and excitation (470 nm) of the fluorescein isothiocyanate(FITC) probe at Lys-501 gave different FITC fluorescene intensity changes at 520 nm in BIPM-FITC doubly labeled anzyme accompanying formatin of reactino intermediates. These data suggest that the fluorescence energy transfer from the BIPM to the FITC probe increased (as follows NaE_1, E_1P,E_2P) and decreased (as follows : E_2P,KE_3, NAE_1). Dynamic fluorescence changes which occurred without phosphorylation or Mg^<2+> seems to reflectchange in the binding states of Na^+ and K^+ or process of the migration of these ions in the pump molecules.
Phopholipase A_2 treatment strongly reduced the fluorescence intensity changes of the BIPM probe with only a slight reductin of the FITC probe in the a-chain of pig kidney Na^+, K^+-ATPase accompanying formatin of phosphoenzymes. The treatment reduced both rate of florescence changes. The anisotropy of both probes were little changed by the treatment. The treatment reduced the Na^+, K^+-ATPase activity of BIPM treated enzyme to 15%. The addition of phosphatidyl serine(PS) or phosphatidyl inositol(PI) increased the extent of the BIPM fluorescence change accompanying the increase in the Na^+, K^+-ATPase activity of BIPM enzyme and the rate of FITC fluorescence changes the BIPM-FITC enzyme. These data suggest that PS or PI which have been shown to be prerequisite for the activity are also prerequisite for the apearance of dynamic B IPM fluorescence change in the viinity of Cys-964 which is supposed to be present in the transmenbrance segment byt not for the FITC fluorescence change in that of Lys-501 to by present in the soluble domain.
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