| Project/Area Number |
05454683
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
神経・脳内生理学
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| Research Institution | Oita Medical University |
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Principal Investigator |
YAMADA Kazuhiro Oita Med.Univ., Physiol., Professor, 医学部, 教授 (20053027)
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| Co-Investigator(Kenkyū-buntansha) |
堀内 桂輔 大分医科大学, 医学部, 助教授 (50183603)
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1993: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Keywords | fluorescence / Mant-ATP / TNP-ADP / active site / myosin heads / contraction / crossbridge / muscle fiber / 筋収縮 / ケージド化合物 / クロスブリッジ / 蛍光ATPアナログ / 収縮蛋白質 / レーザー光 / 螢光ATPアナログ |
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| Research Abstract |
From the measurements in muscle fibers of fluorescence energy transfer between Mant-ATP and TNP-ADP we have found that in addition to the active site TNP nucleotides bind to a secondary site located within 2 nm of the active site of myosin with Kd of approx. 15 muM.In contrast, the fluorescence intensity measurements indicate that Mant-ATP binds only to the active site. The secondary TNP site is closer to the active site than any other site that previously has been fluorescence labelled in the myosin molecule. Thus by using TNP nucleotides bound to the secondary site as a monitor we have detected structural changes in the active site when the myosin heads bind nucleotides and hydrolyse them to generate force.
By pulse-photolysis of caged nucleotides in TNP-ADP labelled fibers in rigor we have established that : (1), on binding ATP and ADP the myosin active site changes its structure considerably ; (2), when the fiber actively contracts, the active site undergoes further structural changes reflecting events at the actin-binding site ; and (3), when the fiber again goes into rigor the fluorescence intensity returns to the previous level in rigor with bound ADP,indicating that during contraction the conformation of the crossbridges is different from that in rigor.
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