| Project/Area Number |
06671626
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Yamagata University School of Medicine |
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Principal Investigator |
KOMIYA Yuichi Yamagata University School of Medicine, Instructor, 医学部, 講師 (70234888)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1995: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | estrogen receptor / positive cooperativity / chemical crosslinking / copper ion / zinc ion / エストロゲンレセプター |
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| Research Abstract |
Interaction of estrogen with unliganded receptor protein dimers : Stabilization of high affinity conformation and a loss of cooperative steroid binding after chemical crosslinking and treatment with Cu (II) ions.
1.The unliganded estrogen receptor proteins were shown to be quantitatively transformed to their dimeric (i.e., 134 kDa), DNA binding configuration ever in the absence of bound ligand. The dimeric structure of the unliganded receptor was demomstrated by chemical crosslinking ; unliganded, DNA-binding receptor proteins, covalently crosslinked without prior ligand binding, were covalently labeled with [^3H] tamoxifen aziridine and evaluated by SDS-polyacrylamide gradient gel electrophoresis after denaturation in the presence and absence of reducing agents.
2.The steroid-binding characteristics of unliganded receptor proteins were evaluated in the presence and absence of receptor-bound DNA.Positive cooperativity (Hill coefficient>1.3) was observed for the unliganded receptor protein at all concentrations above 1 nM.However, after covalent crosslinking with dithiobis (succinimidylpropionate) the cooperative steroid binding was completely eliminated (Hill coefficient=1.07).
3.Similar results were obtained in the presence of 100 muM Cu(II) ions.Zn(II) ions, however, were without effect at equivalent concentrations.
These results demonstrate that estrogen receptor proteins were stable as dimers even in the absence of bound hormone and after covalently chemical crosslinking. The interaction of unliganded estrogen receptor proteins with chemical crosslinking reagents and Cu (II) ions eliminated the cooperative steroid binding. Furthermore, it is suggested that Cu (II) ions influence as one of the regurating factor on cooperative steroid binding with estrogen receptor.
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