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首页> 外文期刊>Biochemistry >LIGHT-DIRECTED GENERATION OF THE ACTIN-ACTIVATED ATPASE ACTIVITY OF CAGED HEAVY MEROMYOSIN
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LIGHT-DIRECTED GENERATION OF THE ACTIN-ACTIVATED ATPASE ACTIVITY OF CAGED HEAVY MEROMYOSIN

机译：笼养重质肌球蛋白的乙酰激活ATPase活性的光导产生。

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An understanding of the molecular mechanism of muscle contraction will require a complete description of the kinetics of the myosin motor in vitro and in vivo. To this end chemical relaxation studies employing light-directed generation of ATP from caged ATP have provided detailed kinetic information in muscle fibers. A more direct approach would be to trigger the actin-activated ATPase activity from a caged myosin, i.e., myosin whose activity is blocked upon derivatization with a photolabile protection group. Herein we report that a new type of caged reagent can be used to prepare a caged heavy meromyosin by modification of critical thiol groups, i.e., a chemically modified motor without activity that can be reactivated at will using a pulse of near-ultraviolet light. Heavy meromyosin modified at Cys-707 with the thiol reactive reagent 1-(bromomethyl)-2-nitro-4,5-dimethoxybenzene does not exhibit an actin-activated ATPase activity and may be viewed as a caged protein. Absorption spectroscopy showed that the thioether bond linking the cage group to Cys-707 is cleaved following irradiation (340-400 nm) via a transient aci-nitro intermediate which has an absorption maximum at 440 nm and decays with a rate constant of 45.6 s(-1). The in vitro motility assay showed that caged heavy meromyosin cannot generate the force necessary to move actin filaments although following irradiation of the image field with a 30 ms pulse of 340-400 nm light the caged group was removed with the concomitant movement of most filaments at a velocity of 0.5-2 mu m/s compared to 3-4 mu m/s for unmodified HMM. The specificity and simplicity of labeling myosin with the caged reagent should prove useful in studies of muscle contraction in vivo.
机译：对肌肉收缩的分子机制的理解将需要完整描述体外和体内肌球蛋白运动的动力学。为此，化学弛豫研究采用了由笼状ATP产生光导的ATP，从而提供了肌肉纤维中的详细动力学信息。一种更直接的方法是从笼中的肌球蛋白即肌球蛋白触发肌动蛋白激活的ATP酶活性，所述肌球蛋白的活性在被光不稳定的保护基团衍生后被阻断。本文中，我们报道了一种新型的笼状试剂，可用于通过修饰关键硫醇基团来制备笼状重肌球蛋白，即无活性的化学修饰的马达，其可以使用近紫外光脉冲随意重新活化。在Cys-707上用硫醇反应试剂1-（溴甲基）-2-硝基-4,​​5-二甲氧基苯修饰的重肌球蛋白不具有肌动蛋白激活的ATPase活性，可以被视为笼状蛋白。吸收光谱表明，在辐射（340-400 nm）之后，通过短暂的ac-硝基中间体裂解连接笼基团与Cys-707的硫醚键，该中间体在440 nm处具有最大吸收并以45.6 s的速率常数衰减（ -1）。体外运动试验表明，笼状沉重的肌球蛋白不能产生移动肌动蛋白丝所需的力，尽管在用340-400 nm的30 ms脉冲照射图像场后，笼状组被移除，同时大多数丝在速度为0.5-2μm / s，而未经修饰的HMM为3-4μm / s。用笼状试剂标记肌球蛋白的特异性和简便性应在体内肌肉收缩研究中被证明是有用的。

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