Ellison Patricia A, DePew Zachary S, Cremo Christine R
Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA.
J Biol Chem. 2003 Feb 14;278(7):4410-5. doi: 10.1074/jbc.M211016200. Epub 2002 Dec 2.
The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240-23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states.
使用三种荧光方法研究了二磷酸腺苷（ADP）和磷酸化对重酶解肌球蛋白肌动蛋白结合特性的影响，这三种方法可监测与芘标记肌动蛋白结合的重酶解肌球蛋白头部数量：（i）ATP诱导解离的幅度；（ii）ADP诱导的芘标记肌动蛋白 - 重酶解肌球蛋白复合物解离的幅度；（iii）重酶解肌球蛋白与芘标记肌动蛋白结合的幅度。无论调节性轻链磷酸化与否或ADP是否存在，两个头部均与芘标记肌动蛋白结合。对于通过用蛋白水解酶消化鸡肫肌球蛋白制备的天然调节型重酶解肌球蛋白，在肌动蛋白结合环处重链裂解5%至95%时均发现了这种行为，表明双头结合是未裂解重链的重酶解肌球蛋白的特性。这些数据与之前使用未裂解的表达制剂的研究结果相反（Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240 - 23245），该研究表明未磷酸化的重酶解肌球蛋白 - ADP复合物的一个头部与肌动蛋白结合，而另一个头部要么不结合要么结合较弱。讨论了两项研究结果差异的可能解释。基于对肌动蛋白 - 重酶解肌球蛋白结合速率常数的分析，我们表明未磷酸化的重酶解肌球蛋白在ADP存在下似乎呈现一种特殊状态。数据表明，在ADP存在下，一个头部快速结合，第二个头部结合较慢。在所有其他状态下，两个头部以相同速率与肌动蛋白结合。
