Analysis of the Ca^<2+> release mechanism from the E2PCa intermediate of sarcoplasmic reticulum Ca^<2+> pump
| Project/Area Number |
18570119
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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 | Asahikawa Medical College |
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Principal Investigator |
YAMASAKI Kazuo Asahikawa Medical College, School of Medicine, Assistant Professor (60241428)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,890,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Sarcoplasmic reticulum / Calcium ion / Ca^<2+> pump / Enzyme Kinetics / SERCA1a / SERCA1a |
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| Research Abstract |
Development of method for measurement of Ca^<2+> binding to mutants of Ca^<2+> -ATPase, and improvement of its accuracy
Measurement of bound Ca^<2+> was enabled to be done directly by devising the washing solution of the membrane filtoration method in this research, though it was very difficult in the analysis that used the mutant of sarcoplasmic reticulum Ca^<2+>-ATPase to measure Ca^<2+> binding from the low degree of the amount of appearance so far.
Analysis of Ca^<2+> releasing process that uses mutants
In mutant that changes Tyr^<122> of sarcoplasmic reticulum Ca^<2+>-ATPase into Ala (Y122A), I have already found that the E2PCa intermediate accumulates during it in the steady state. In crystal structures of E2P analogs, Tyr^<122> located at the center of the hydrophobic interaction network (Tyr^<122> Hydrophobic cluster: Y122-HC) which situated between cytoplasm domains. The Ala substitution mutants of the other residues include in Y122-HC were made and analyzed its kinetics of Ca^<2
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+> releasing step. It was revealed from this analysis that the rates of Ca^<2+> release toward lumen side and of association had decreased remarkably compared with a wild type in each mutant. Moreover, the accumulation of the E2PCa in the steady state was observed in a part of mutants as well as Y122A. When Ca^<2+> release from the phosphorylation medium was measured directly by using the above-mentioned method about L119A in this mutant, it was shown that Ca^<2+> release from E2PCa^<2+> was obviously slower than the conversion of the phosphoenzyme (E1PCa_2⇒E2PCa_2). The role of Y122-HC in Ca^<2+> transport of Ca^<2+>-ATPase became clear from the above-mentioned result.
Analysis that uses wild type Ca^<2+>-ATPase
In a series of analysis, it was newly clarified to wild type Ca^<2+>-ATPase that the character to Ca^<2+> of E2P resembled the Y122-HC mutant very much in the absence of K^+. The result that K^+ ion plays an important role to Ca^<2+> release process of Ca^<2+>-ATPase is a finding of an important for recognize the Ca^<2+> transport mechanism of Ca^<2+>-ATPase. Less
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Report
(3 results)
Research Products
(19 results)
