Thin filament activation probed by fluorescence of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle
Thin filament activation probed by fluorescence of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle
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DOI：
10.1016/s0006-3495(99)77102-x
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发表时间：
1999-11-01
影响因子：
3.4
通讯作者：
Chalovich, JM
中科院分区：
文献类型：
--
作者：
Brenner, B;Kraft, T;Chalovich, JM
描述了一种将单个兔腰肌纤维的天然肌钙蛋白交换为外部应用的肌钙蛋白复合物的方法，而不会检测到对带皮纤维的功能特性的损害。该方法用于将天然肌钙蛋白交换为兔骨骼肌钙蛋白，肌钙蛋白 I 亚基的 Cys(133) 上带有荧光标记 (N-((2-(碘乙酰氧基)乙基)-N-甲基) 氨基-7-硝基苯甲-2-氧杂-1,3-二唑, IANBD)。 IANBD 标记的肌钙蛋白 I 先前已在溶液研究中用作重建肌动蛋白丝激活状态的指示剂（Trybus 和 Taylor，1980，Proc. Natl Acad. Sci. USA. 77：7209-7213）。在带皮纤维中，当处于弱结合状态的横桥附着到肌动蛋白丝上时，该探针的荧光不受影响，但在添加 Ca2+ 时或当处于强结合状态的横桥附着到肌动蛋白上时，荧光会减弱。当 Ca2+ 升高至饱和浓度时，观察到最大程度的减少。在强结合状态下额外连接交叉桥不会进一步减少荧光。单独以强结合状态（低 Ca2+ 浓度）连接桥的效果仅是添加钙时所见最大减少量的一半左右。这说明 IANBD 标记的肌钙蛋白 I 的荧光可用于评估细丝激活，如之前在溶液研究中介绍的那样。另外，在非饱和Ca2+浓度下； IANBD 荧光可用于将肌球蛋白头的状态直接分类为弱结合（非激活）和强结合（激活），而与离子强度或其他实验条件无关。此外，这里提出的方法不仅可以用作将天然骨骼肌钙蛋白及其亚基交换为各种荧光标记或突变肌钙蛋白亚基的手段，而且还允许将天然骨骼肌钙蛋白交换为心肌肌钙蛋白。
A method is described for the exchange of native troponin of single rabbit psoas muscle fibers for externally applied troponin complexes without detectable impairment of functional properties of the skinned fibers. This approach is used to exchange native troponin for rabbit skeletal troponin with a fluorescent label (N-((2-(iodoacetoxy)ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-1,3-diazole, IANBD) on Cys(133) of the troponin I subunit. IANBD-labeled troponin I has previously been used in solution studies as an indicator for the state of activation of reconstituted actin filaments (Trybus and Taylor, 1980, Proc. Natl Acad. Sci. USA. 77:7209-7213). In the skinned fibers, the fluorescence of this probe is unaffected when cross-bridges in their weak binding states attach to actin filaments but decreases either upon the addition of Ca2+ or when cross-bridges in their strong binding states attach to actin. Maximum reduction is observed when Ca2+ is raised to saturating concentrations. Additional attachment of cross-bridges in strong binding states gives no further reduction of fluorescence. Attachment of cross-bridges in strong binding states alone (low Ca2+ concentration) gives only about half of the maximum reduction seen with the addition of calcium. This illustrates that fluorescence of IANBD-labeled troponin I can be used to evaluate thin filament activation, as previously introduced for solution studies. In addition, at nonsaturating Ca2+ concen; trations IANBD fluorescence can be used for straightforward classification of states of the myosin head as weak binding (nonactivating) and strong binding (activating), irrespective of ionic strength or other experimental conditions. Furthermore, the approach presented here not only can be used as a means of exchanging native skeletal troponin and its subunits for a variety of fluorescently labeled or mutant troponin subunits, but also allows the exchange of native skeletal troponin for cardiac troponin.