Gordon A M, Ridgway E B, Yates L D, Allen T
Department of Physiology and Biophysics, University of Washington, Seattle 98195.
Adv Exp Med Biol. 1988;226:89-99.
Data from intact and skinned muscle fibers support the hypothesis that cross-bridge interaction modifies TnC structure and calcium activation. Barnacle single muscle fibers microinjected with the calcium bioluminescent photoprotein, aequorin, show extra light (calcium) when shortened during the declining phase of the calcium transient. The extra calcium is increased by increases in muscle force, and its decline is delayed at higher forces. This extra calcium occurs probably because calcium binding to the activating sites is increased by cross-bridge interaction. In rabbit muscle, TnC structure is modified by cross-bridge interaction, since in skinned rabbit psoas muscle fibers TnC extraction is slower at shorter sarcomere lengths, where cross-bridge attachment is increased. Thus the rigor bridges formed in the extraction solution strengthen the attachment of TnC to the thin filament. Reintroduction of TnC, labeled with fluorescent probes near the Ca specific binding sites (Danzylaziridine-DANZ) and Ca-Mg sites (Rhodamine), into the partially TnC extracted fibers allows us to assess the structural changes (total fluorescence for the DANZ probes, linear dichroism for the RHOD probe) in response to calcium binding and cross-bridge attachment. At sarcomere lengths beyond overlap, calcium binding increases the DANZ-TnC fluorescence and disorders the RHOD-TnC label. At full overlap of filaments, rigor cross-bridges also increase the DANZ-TnC fluorescence and RHOD-TnC disorder. The addition of calcium in rigor increases the DANZ-TnC fluorescence little but causes additional RHOD-TnC disorder, although both fluorescence and disorder are increased further in the presence of calcium plus MgATP. In fibers containing DANZ-TnC, decreasing MgATP in the absence of calcium increases both the force and the fluorescence as rigor cross-bridges activate the muscle. In the presence of calcium, an increase in MgATP to 0.75 microM produces a small fluorescent enhancement, but an increase in MgATP to 10 microM and to 3 mM produces a substantial enhancement. The data imply that calcium activates the thin filament, but that the filament is activated further by rigor cross-bridges. Active cross-bridges activate the thin filament still further. Thus, cross-bridges modify TnC structure and calcium activation, with active cross-bridges being more effective than rigor cross-bridges.
完整和去皮肌纤维的数据支持这样一种假说，即横桥相互作用会改变肌钙蛋白C（TnC）的结构和钙激活。用钙生物发光光蛋白水母发光蛋白微量注射藤壶单肌纤维，在钙瞬变的下降阶段缩短时会显示额外的光（钙）。额外的钙会随着肌肉力量的增加而增加，并且在更高的力量下其下降会延迟。这种额外的钙可能是因为横桥相互作用增加了钙与激活位点的结合。在兔肌肉中，横桥相互作用会改变TnC的结构，因为在去皮的兔腰大肌纤维中，在较短的肌节长度下TnC的提取较慢，而在这些长度下横桥附着增加。因此，在提取溶液中形成的强直桥会加强TnC与细肌丝的附着。将在钙特异性结合位点（丹酰氮丙啶-DANZ）和钙-镁位点（罗丹明）附近用荧光探针标记的TnC重新引入部分提取了TnC的纤维中，使我们能够评估响应钙结合和横桥附着的结构变化（DANZ探针的总荧光，RHOD探针的线性二色性）。在肌节长度超过重叠时，钙结合会增加DANZ-TnC荧光并使RHOD-TnC标记无序化。在细丝完全重叠时，强直横桥也会增加DANZ-TnC荧光和RHOD-TnC无序化。在强直状态下添加钙只会使DANZ-TnC荧光略有增加，但会导致RHOD-TnC进一步无序化，尽管在存在钙加MgATP的情况下荧光和无序化都会进一步增加。在含有DANZ-TnC的纤维中，在没有钙的情况下降低MgATP会随着强直横桥激活肌肉而增加力量和荧光。在有钙的情况下，将MgATP增加到0.75微摩尔会产生小的荧光增强，但将MgATP增加到10微摩尔和3毫摩尔会产生显著增强。数据表明钙激活细肌丝，但细肌丝会被强直横桥进一步激活。活性横桥会使细肌丝进一步激活。因此，横桥会改变TnC结构和钙激活，活性横桥比强直横桥更有效。
