Labeling of myosin with a fluorescent analog of ATP and the fluorescent and mechanical transients after photolysis of caged nucleotides in skeletal muscle fibers.

用 ATP 荧光类似物标记肌球蛋白，以及骨骼肌纤维中笼状核苷酸光解后的荧光和机械瞬态。

• 批准号: 05454683
• 负责人: YAMADA Kazuhiro
• 金额: $ 4.42万
• 依托单位: Oita Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454683/
• 关键词: 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

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.

从Mant-ATP和TNP-ADP之间的荧光能量转移的肌纤维中的测量中，我们发现，除了活性位点之外，TNP核苷酸结合到位于肌球蛋白活性位点的2nm内的第二位点，Kd约为1.5nm。相反，荧光强度测量表明Mant-ATP仅与活性位点结合。次级TNP位点比以前在肌球蛋白分子中被荧光标记的任何其他位点更接近活性位点。因此，我们用TNP核苷酸作为监测物，检测了肌球蛋白头部与核苷酸结合并水解产生力时活性部位的结构变化，用脉冲光解TNP-ADP标记的肌纤维中的笼状核苷酸，证实了：（1）肌球蛋白活性部位与ATP和ADP结合时，其结构发生了显著变化;（2）当纤维主动收缩时，活性位点经历进一步的结构变化，反映肌动蛋白结合位点的事件;（3）当纤维再次进入僵直状态时，荧光强度恢复到先前与ADP结合的僵直水平，表明在收缩期间横桥的构象与僵直时不同。

项目成果

Keisuke Horiuti: "Transient contraction of Muscle fibers on photorelease of ATP at intermediate dconcentrations of Ca2+." Biophysical Journal. 67. 1925-1932 (1994)

Keisuke Horiuti：“在中等浓度的 Ca2 下，ATP 光释放时肌肉纤维的瞬时收缩。”

Yumiko Emoto: "Tension relaxation induced by pulse photolysis of caged ATP in partially crosslinked fibers from rabbit psoas muscle." Proc.Natl.Acad.Sci.USA. (in press). (1995)

Yumiko Emoto：“兔腰肌部分交联纤维中笼中 ATP 的脉冲光解引起的张力松弛。”

Kazuhiro Yamada: "The conformation changes of the active site of myosin associated with the ATP binding in skinned skeletal muscle fibres" J.Muscle Res.and Cell Motil.(in press). (1995)

Kazuhiro Yamada：“与带皮骨骼肌纤维中 ATP 结合相关的肌球蛋白活性位点的构象变化”J.Muscle Res.and Cell Motil.（出版中）。

Yamada,K.: "Labeling of myosin with a fluorescent analog of ATP and the fluorescent transients after photolysis caged nucleotides in skeletal muscle fibers." Biophysical Journal. 68. 331s (1995)

Yamada,K.：“用 ATP 荧光类似物标记肌球蛋白，以及骨骼肌纤维中光解笼中核苷酸后的荧光瞬变。”

Kazuhiro Yamda: "Labeling of myosin with a fluorescent analog of ATP and the fluorescence transients after photolysis of caged nucleotides in skeletal muscle fibers." Biophysical Journal. (in press). (1995)

Kazuhiro Yamda：“用 ATP 荧光类似物标记肌球蛋白，以及骨骼肌纤维中笼状核苷酸光解后的荧光瞬变。”