Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils
Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils
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DOI：
10.1016/s0006-3495(00)76698-7
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发表时间：
2000-03-01
影响因子：
3.4
通讯作者：
Berger, CL
中科院分区：
文献类型：
--
作者：
Cooper, WC;Chrin, LR;Berger, CL
：肌球蛋白亚段1(S1)可以用荧光探针FHS(6-[荧光素-5(和6)-氨基]己酸琥珀酰亚胺酯)(Bertrand，R，J.Derancourt和R.Kosa)在Lys-553处进行特异性修饰。1995年。生物化学。34.9500-9507)，并且已经证明在低离子强度下，用碘对FHS-S1的溶剂猝灭对肌动蛋白结合很敏感(MacLean，Chrin和Berger，2000年)。生物物理学，J.000-000)。为了扩展这些结果并检测在钙激活过程中肌丝晶格中肌动蛋白结合的肌球蛋白头部的比例，我们对骨骼肌肌原纤维进行了修饰，并与eDC(1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide)温和地交联以防止缩短，通过FHS，肌球蛋白重链似乎是主要的标记位置，并且碘化物猝灭模式与获得的肌球蛋白SI在溶液中的结果一致，这表明LYS-553FHS确实是FHS掺入骨骼肌肌纤维的主要位置。钙激活的FHS-肌原纤维的碘猝灭结果表明，在等长收缩过程中，29%的肌球蛋白头部在低离子强度下与肌丝晶格中的肌动蛋白强烈结合。这些结果表明，在更复杂和与生理相关的制备方法中，FHS可以特异性地修饰肌球蛋白，从而可以在体外运动试验中以及在单个肌肉纤维内的等长和等张收缩过程中实时检测与肌动蛋白的跨桥相互作用。
:Myosin subfragment 1 (S1) can be specifically modified at Lys-553 with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) (Bertrand, R,, J. Derancourt, and R. Kassab. 1995. Biochemistry. 34.9500-9507), and solvent quenching of FHS-S1 with iodide has been shown to be sensitive to actin binding at low ionic strength (MacLean, Chrin, and Berger, 2000. Biophys, J. 000- 000). In order to extend these results and examine the fraction of actin-bound myosin heads within the myofilament lattice during calcium activation, we have modified skeletal muscle myofibrils, mildly cross-linked with EDC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) to prevent shortening, with FHS, The myosin heavy chain appears to be the predominant site of labeling, and the iodide quenching patterns are consistent with those obtained for myosin SI in solution, suggesting that Lys-553 is indeed the primary site of FHS incorporation in skeletal muscle myofibrils. The iodide quenching results from calcium-activated FHS-myofibrils indicate that during isometric contraction 29% of the myosin heads are strongly bound to actin within the myofilament lattice at low ionic strength., These results suggest that myosin can be specifically modified with FHS in more complex and physiologically relevant preparations, allowing the real time examination of cross-bridge interactions with actin in in vitro motility assays and during isometric and isotonic contractions within single muscle fibers.