Ueno H, Harrington W F
J Mol Biol. 1986 Jul 5;190(1):59-68. doi: 10.1016/0022-2836(86)90075-6.
We have used alpha-chymotrypsin as an enzyme-probe to detect local melting in the subfragment-2 region of the cross-bridges of rigor myofibrils and glycerinated psoas fibers. The kinetics of proteolysis and the sites of cleavage were determined at various temperatures over the range 5 to 40 degrees C by following the decay of the myosin heavy chain and the rates of appearance of light meromyosin fragments, using electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. Cleavage occurs primarily at the 72,000 Mr and 64,000 Mr (per polypeptide chain from the C terminus of myosin) sites within the light meromyosin-heavy meromyosin hinge domain of the subfragment-2 region, under all experimental conditions. At pH 8.2 to 8.3 and at low divalent metal ion (0.1 mM), where the actin-bound cross-bridges are thought to be released from the thick filament surface, the intrinsic cleavage rate constant (k) increases markedly as the temperature is raised. This suggests substantial thermal destabilization of the released cross-bridge in the intact contractile apparatus. Addition of divalent metal ion (10 mM) lowers the cleavage rate and shifts the k versus temperature profile to higher temperatures. Normalized rate constants for chymotryptic cleavage within the subfragment-2 hinge region of released cross-bridges (pH 8.2, low divalent metal) of rigor fibers were markedly lower than activated fibers at all temperatures investigated (5 to 40 degrees C). Results show that conformational melting within the subfragment-2 hinge region is amplified on activation and is well above that observed when the actin-attached rigor bridge is passively released from the thick filament surface.
我们使用α-胰凝乳蛋白酶作为酶探针，来检测僵直肌原纤维和甘油化腰大肌纤维横桥亚片段-2区域的局部解链情况。通过在含十二烷基硫酸钠的聚丙烯酰胺凝胶上进行电泳，跟踪肌球蛋白重链的衰减以及轻酶解肌球蛋白片段出现的速率，在5至40摄氏度的不同温度下测定了蛋白水解的动力学和裂解位点。在所有实验条件下，裂解主要发生在亚片段-2区域轻酶解肌球蛋白-重酶解肌球蛋白铰链结构域内分子量为72,000和64,000（每条多肽链从肌球蛋白C末端起算）的位点。在pH 8.2至8.3以及低二价金属离子（0.1 mM）的条件下，肌动蛋白结合的横桥被认为从粗肌丝表面释放，随着温度升高，固有裂解速率常数（k）显著增加。这表明在完整收缩装置中，释放的横桥存在明显的热不稳定现象。添加二价金属离子（10 mM）会降低裂解速率，并将k与温度的关系曲线向更高温度偏移。在所有研究温度（5至40摄氏度）下，僵直纤维释放的横桥（pH 8.2，低二价金属）亚片段-2铰链区域内胰凝乳蛋白酶裂解的标准化速率常数明显低于活化纤维。结果表明，亚片段-2铰链区域内的构象解链在活化时会被放大，且远高于肌动蛋白附着的僵直横桥从粗肌丝表面被动释放时所观察到的情况。
