| Project/Area Number |
15570124
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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 | Rikkyo University |
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Principal Investigator |
MAKINO Ryu Rikkyo University, College of Science, Professor, 理学部, 教授 (40101026)
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| Co-Investigator(Kenkyū-buntansha) |
HORI Hiroshi Osaka University, Graduate School of Engineering Science, Associate Professor, 基礎工学部, 助教授 (20127294)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2005: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | guanylate cyclase / nitric oxide / YC-1 / BAY 41-2272 / nucleotide / carbon monoxide / 一酸化窒素(NO) / 一酸化炭素 |
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| Research Abstract |
Soluble guanylate cyclase is a heterodimeric hemoprotein composed of α- and β-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide binding sites with high and low affinity for GMP-CPP (α,β-methylene GTP). In contrast to GMP-CPP binding, 2'-dADP occupied both sites with equivalent affinities. AMP-PNP (β,γ-imido ATP) which competitively inhibited the cyclase reaction, bound solely to the high-affinity site, indicating the role of this site as catalytic site. The function of the low-affinity site was examined using allosteric activators, YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2'-dADP, probably by competing for the same site as 2'-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2'-dADP as well as GMP-CPP. These suggest that the activators compete with these nucleotides for the low-affinity site. Infrared and EPR analyses of the enzymic CO- and NO-hemes also supported the suggested role of the low-affinity site as a target for the activators. Our results imply that the low-affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.
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