Molecular Mechanism of Energy Transduction and Regulation in Na^+ pump and K^+ pump
Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||Hokkaido University|
TANIGUCHI Kazuya Hokkaido University, 理学部, 教授 (40028204)
BEECHEM J. M. Vanderbilt University, 医学部, 准助教授
MARDH Sven Uppsala University, 医学部・生理化学部, 准助教授
SVEN Mardh ウプサラ大学, 医学部・生理化学部, 准助教授
MARD Sven ウプサラ大学, 医学生理学部, 准教授
|Project Period (FY)
1989 – 1991
Completed(Fiscal Year 1991)
|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1991 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1990 : ¥1,000,000 (Direct Cost : ¥1,000,000)
|Keywords||Na^+ / K^+-ATPase / H^+ / -K^+-ATPase / Transport ATPase / ケイ光プロ-ブ / リン酸化 / 構造変化|
Energy Transduction and Regulation in Molecular Mechanism of Na^+ pump and K^+ pump
1. Studies of Fluorescence energy transfer between labeled probes in Na^+ pump were made during sequential appearance of reaction intermediates. The data clearly showed that the fluorescence energy transfer from BIPM probe (Cys 964) to FITC probe (Lys 501) increased accompanying formation of E_2P from NaE_1 via E_1P, and decreased accompanying formation of NaE_1 from E_2P via KE_2. The anisotropy of both probes changed little accompanying formation of these intermediates. Opposite change in fluorescence energy transfer from BIPM to ANM probes are now detected. The estimation of the distances between those probes are in progress.
2. Relationships between conformational changes and phosphorylation-dephosphosphorylation cycles in Na^+ pump and K^+ pump were compared. The data show that the conformational changes and the phosphorylation-dephosphorylation cycle is not tightly coupled and strongly suggest that
the conformational changes reflect the migration of Na^+ and K^+ in the Na^+ pump molecule. To clarify the mechanism of energy transduction, to quantify the migration of those ions seems to be indispensable.
Conformational changes in H^+ pump were studied using intrinsic (tryptophan) and extrinsic (BIPM, FITC) fluorescence probes. Fluorescence intensity of Trp residues increases after formation of phosphoenzymes. Addition of K^+ to the phosphoenzyme reduced the fluorescence to the same level to that of HE_1. Fluorescence intensity of BIPM probe decreased with nearly the same rate constant of phosphorylation. Addition of K^+ to the phosphoenzyme slightly increased the BIPM fluorescence. These data clearly show that both fluorescence probes sense different enzyme states as already shown in Na^+ pump using various fluorescence probes.
3. Na^+ pump was modified to label the fluorescence probe near ATP binding domain with pyridoxal phosphate in the presence of Na^+ or K^+. Na^+, K^+-ATPase activity and phosphorylation from ATP were inhibited while phosphorylation from acetyl phosphate remained. The sites of pyridoxal phosphate probe attached seemed to be Lys-664 in NaE_1 and both Lys-664 and Lys-469 in KE_2.
4. Role of phospholipids in the conformational change in Na^+, K^+-ATPase were studied. The results showed that the change in the fluorescence intensity of BIPM probe at Cys-964 accompanying accumulation of E_2P is more susceptible than that of E_1P to the phospholipase A treatment.
5. Cyclic AMP-dependent protein kinase was purified from pig kidney red outer medulla from which Na^+, K^+-ATPase has been purified. Na^+, K^+-ATPase was phosphorylated by the purified kinase. One mole of phosphate was detected in the one mole of alpha-chain sequence for the site of phosphorylation and the change in the Na^+, K^+-ATPase activity and the affinity for ligands after phosphorylation by the kinase are now in progress. Less
Research Products (31results)