A STRUCTURAL AND KINETIC-STUDY ON MYOFIBRILS PREVENTED FROM SHORTENING BY CHEMICAL CROSS-LINKING
A STRUCTURAL AND KINETIC-STUDY ON MYOFIBRILS PREVENTED FROM SHORTENING BY CHEMICAL CROSS-LINKING
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DOI：
10.1021/bi00079a023
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发表时间：
1993-07-20
期刊：
影响因子：
2.9
通讯作者：
BARMAN, T
中科院分区：
文献类型：
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作者：
HERRMANN, C;SLEEP, J;BARMAN, T
在先前的工作中，我们研究了Ca 2+激活的肌原纤维的Mg 2 +-ATP酶活性的早期步骤[Houadjeto，M.，Travers，F.，& Barman，T.（1992）Biochemistry 31，1564-1569]。肌原纤维自由收缩，并且所获得的结果是指在没有外部负荷的情况下肌原纤维收缩的ATP酶循环。在这里，我们研究了肌原纤维等长收缩的ATP酶。为了防止缩短，我们用1-乙基-3-[3-（二甲氨基）丙基]碳二亚胺（EDC）交联它们。SDS-PAGE和Western blot分析表明，肌球蛋白杆广泛交联，8%的肌球蛋白头交联到细丝。在0.1 M醋酸钾，pH 7.4和4 ℃，通过快速流动淬火法的交联肌原纤维的瞬态动力学进行了研究。ATP结合步骤用冷ATP追踪法和P（i）猝发法研究。在P（i）爆发实验中，对于交联（等长）和未交联（等张）肌原纤维，爆发的大小在实验误差内等于ATP酶位点浓度（由冷ATP追踪法测定）。这表明，在这两种情况下，限速步骤都是在ATP裂解之后。当交联时，Ca 2+激活的肌原纤维的k（cat）从1.7降低到0.8 s-1。这与中等速度下缩短的纤维比等长纤维具有更高的ATP酶活性的观察结果一致。在松弛条件下（-Ca 2+），k（猫）仍然很大（0.6 s-1），大概是由于8%的头部交联到细丝引起的僵硬激活。
In previous work, we studied the early steps of the Mg2+-ATPase activity of Ca2+-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T.(1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4-degrees-C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the P(i) burst method. In P(i) burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the k(cat) of Ca2+-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers. Under relaxing conditions (-Ca2+), the k(cat) remains large (0.6 s-1) presumably due to rigor activation induced by the 8% heads cross-linked to the thin filaments.