Muhlrad A
Cardiovascular Research Institute, University of California, San Francisco.
Biochim Biophys Acta. 1991 Apr 29;1077(3):308-15. doi: 10.1016/0167-4838(91)90545-b.
Recently we reported that the isolated 23 kDa N-terminal fragment of myosin heavy chain, which contains the 'consensus' ATP binding site, binds to actin in an ATP-sensitive manner (Muhlrad, A. (1989) Biochemistry 28, 4002). In order to determine whether the 'consensus' ATP site has a role in the ATP-dependent actin binding of the fragment, we isolated a shorter 21 kDa N-terminal fragment, which contains only a part of the 'consensus' site. The 21 kDa fragment was obtained by photocleavage of myosin subfragment-1 in the presence of vanadate (Mocz, G. (1989) Eur. J. Biochem. 179, 373); the cleavage was followed by dissociation of the S-1 heavy chain fragments with guanidine hydrochloride and renaturation. The isolated 21 kDa fragment binds to F-actin, since it cosediments with actin, inhibits the actin-activated ATPase activity of myosin subfragment-1 and shows increase in light scattering upon titration by actin. The affinity of the binding is rather high (Kassoc = 0.83.10(7) M-1). The light scattering increase is reversed, e.g., the 21 kDa-actin complex is dissociated, upon addition of ATP both in the presence and absence of Mg, but less ATP is needed for dissociation when Mg is absent. Other polyphosphates, including inorganic triphosphate, pyrophosphate and ADP, also dissociate both the 21 kDa-actin and 23 kDa-actin complexes but the latter needs a higher concentration of polyphosphates for dissociation. However, these polyphosphates, except ATP, do not dissociate the (subfragment-1)-actin complex in the absence of Mg. The 21 kDa-actin and the 23 kDa-actin complexes are also dissociated by increasing ionic strength or by a low concentration of polyglutamate, which hardly affect the light scattering of the (subfragment-1)-actin complex. The results indicate that the binding of the N-terminal fragments of myosin to actin, unlike that of intact subfragment-1, is essentially of electrostatic nature. The polyanions dissociate the myosin fragment-actin complexes not by reacting with the 'consensus' ATP binding site, but by competing with actin for a positively charged binding site on the 21 kDa fragment. The only positively charged cluster in the amino acid sequence of this fragment is the 143-147 stretch, which may participate in forming the actin binding site.
最近我们报道，分离得到的肌球蛋白重链23 kDa N端片段含有“共有”ATP结合位点，它以ATP敏感的方式与肌动蛋白结合（穆尔拉德，A.（1989年）《生物化学》28卷，4002页）。为了确定“共有”ATP位点是否在该片段依赖ATP的肌动蛋白结合中起作用，我们分离出了一个更短的21 kDa N端片段，它只包含“共有”位点的一部分。21 kDa片段是通过在钒酸盐存在下对肌球蛋白亚片段-1进行光裂解得到的（莫茨，G.（1989年）《欧洲生物化学杂志》179卷，373页）；裂解后用盐酸胍使S-1重链片段解离并复性。分离得到的21 kDa片段与F-肌动蛋白结合，因为它与肌动蛋白一起沉降，抑制肌球蛋白亚片段-1的肌动蛋白激活的ATP酶活性，并且在肌动蛋白滴定后光散射增加。结合亲和力相当高（Kassoc = 0.83×10⁷ M⁻¹）。无论是在有镁还是无镁的情况下，加入ATP后光散射增加都会逆转，例如21 kDa-肌动蛋白复合物会解离，但无镁时解离所需的ATP较少。其他多磷酸盐，包括无机三磷酸、焦磷酸和ADP，也会使21 kDa-肌动蛋白和23 kDa-肌动蛋白复合物解离，但后者解离需要更高浓度的多磷酸盐。然而，除了ATP外，这些多磷酸盐在无镁的情况下不会使（亚片段-1）-肌动蛋白复合物解离。增加离子强度或低浓度的聚谷氨酸也会使21 kDa-肌动蛋白和23 kDa-肌动蛋白复合物解离，而这几乎不影响（亚片段-1）-肌动蛋白复合物的光散射。结果表明，肌球蛋白N端片段与肌动蛋白的结合，与完整的亚片段-1不同，本质上是静电性质的。多阴离子使肌球蛋白片段-肌动蛋白复合物解离，不是通过与“共有”ATP结合位点反应，而是通过与肌动蛋白竞争21 kDa片段上的一个带正电的结合位点。该片段氨基酸序列中唯一的带正电簇是143 - 147区段，它可能参与形成肌动蛋白结合位点。
