2000 Fiscal Year Final Research Report Summary
Subunit interaction of Sodium Pump
| Project/Area Number |
11680620
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KAYA Shunji Hokkaido Univ. Grad. School of Sci., Asso. Prof., 大学院・理学研究科, 助教授 (90186023)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Sodium pump / fluorescent probe / ion occlusion |
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| Research Abstract |
Based on the kinetic investigation of the formation and decay of phosphoenzyme intermediate Na/K-ATPase labeled with several fluorescent probes, we have found that both BIPM and RH-421 probes changes their fluorescence intensity depending on the accumulating intermediates. The data suggest that each probe can sense not the formation of phosphoenzyme itself but the binding state of sodium or potassiumions. Fluorescence changes of each probe did not reflect precisely the same molecular events.
The amount of ATP (or phosphate) bound to the enzyme during ATP hydrolysis has been measured. Furthermore, we have compared the amount of occluded Rb in the presence of various combination of ligands. Na/K-ATPase has been shown to bind 1 and 0.5 mol 32P/mol a-subunit in the presence [a-32P]ATP and [g-32P]ATP, respectively, accompanied by a maximum accumulation of 0.5 mol of phosphoenzyme. The enzyme occluded a maximum of 2 mol of Rb/mol of a-subunit. The addition of Na, Mg and ATP induced 1 mol 86Rb/ mol a-subunit with no detectable phosphoenzyme. 0.5 mol of ATP can still bind to Rb occluded enzyme. Electron microscopy of rotary shadowed solubilized Na/K-ATPases and an antibody-Na/K-ATPase complex indicated the presence of tetraprotomeric structures (ab) 4. These data suggest that Na/K- dependent ATP hydrolysis occur via the sequential appearance of (NaE1 P : EATP) 2, (E2P : EATP) 2 and (KE2 : EATP) 2, each of which has been previously referred to as NaE1P, E2P and KE2 in Post-Albers mechanism. We propose a tetramer mechanism for ATP hydrolysis in Na/K-ATPase.
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Research Products
(16 results)
