Duong A M, Reisler E
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Biochemistry. 1989 Feb 7;28(3):1307-13. doi: 10.1021/bi00429a054.
Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.
在ATP水解过程中测量肌原纤维中横桥与肌动蛋白的结合，需要事先固定肌原纤维以防止其收缩。在严格条件下于4℃使用10 mM碳二亚胺（EDC）可实现肌原纤维的最佳交联。固定后的肌原纤维MgATP酶活性升高（150%）且无法收缩。通过胰凝乳蛋白酶消化及随后的SDS凝胶电泳分析判断，这些肌原纤维中不到25%的肌球蛋白头部发生了交联。分离得到的未交联肌球蛋白头部显示出与标准完整S-1类似的pH依赖性Ca2+和EDTA（K+）-ATP酶活性。为了测量肌球蛋白与肌动蛋白的结合，在严格、松弛和活性状态条件下，将修饰后的肌原纤维以1:50的重量比用胰蛋白酶消化。然后用胰凝乳蛋白酶切割胰蛋白酶消化反应的等分试样，以产生分离的肌球蛋白头部及其片段。通过分析SDS凝胶上肌球蛋白重链条带的衰减，得出了所有条件下肌球蛋白的切割速率，并能够测量在MgATP存在下肌原纤维中肌动球蛋白的结合。使用这种方法，我们检测到在ATP水解过程中，肌原纤维中25±6%的肌球蛋白头部与肌动蛋白存在类似僵直的结合。
