ANALYSIS OF THE INTERACTIONS OF ACTIN DEPOLYMERIZING FACTOR WITH G-ACTIN AND F-ACTIN
ANALYSIS OF THE INTERACTIONS OF ACTIN DEPOLYMERIZING FACTOR WITH G-ACTIN AND F-ACTIN
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DOI：
10.1021/bi00089a015
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发表时间：
1993-09-28
期刊：
影响因子：
2.9
通讯作者：
BAMBURG, JR
中科院分区：
文献类型：
--
作者：
HAYDEN, SM;MILLER, PS;BAMBURG, JR
鸡肌动蛋白解聚因子（ADF）是一种肌动蛋白结合蛋白，在体外能迅速解聚肌动蛋白丝，产生1：1的ADF和肌动蛋白单体复合物。在这里，我们表明，ADF保护肌动蛋白单体变性EDTA通过抑制肌动蛋白结合核苷酸的交换。在低离子强度条件下，由核苷酸（1，N6-乙烯基-ATP）交换测定的ADF-肌动蛋白复合物的近似解离常数（K（D））约为150 nM，且不依赖于钙。此外，ADF单体肌动蛋白抑制肌动蛋白组装以及ATP水解，通常伴随着大会。复合物的形成被证明之间的ADF和肌动蛋白含有ATP，ADP，或AMPPNP作为绑定的核苷酸。ADF-ATP-肌动蛋白复合物和ADF-AMPPNP-肌动蛋白复合物的K（D）均计算为0.1-0.2 μ M，而ADF-ADP-肌动蛋白复合物的K（D）约为1.3 μ M。ADF可以与F-肌动蛋白以化学计量的方式共沉淀或共沉淀，但这些相互作用是pH依赖的。在6.5和7.1之间的pH值，ADF与F-肌动蛋白共沉淀，并表现出只有弱解聚活性。ADF结合是协同的，并且在ADF：肌动蛋白摩尔比为1：1时饱和。在7.1和7.7之间的pH下，ADF显示出增加的解聚活性和较少的F-肌动蛋白结合。在pH 8.0时，ADF以化学计量方式解聚F-肌动蛋白。鬼笔环肽抑制ADF的F-肌动蛋白结合和解聚活性。在pH 8.0，亚化学计量量的ADF，但不是ADF-肌动蛋白复合物，瞬时增强成核能力的F-肌动蛋白的解决方案中的钙不敏感的方式，这表明ADF具有弱切断活性，但不盖的方式，抑制恢复原来的细丝数量的细丝末端。我们得出结论，从这些结果，ADF是一个noncapping，钙离子不依赖性，pH敏感的F-肌动蛋白结合/切断蛋白，能够形成中等亲和力的复合物与肌动蛋白单体，其中核苷酸交换被抑制。这些研究结果的意义，一个假定的细胞ADF的作用进行了讨论。
Chick actin depolymerizing factor (ADF) is an actin binding protein previously shown to rapidly depolymerize actin filaments in vitro, yielding a 1:1 complex of ADF and actin monomer. Here we show that ADF protects actin monomer from denaturation by EDTA by inhibiting the exchange of actin-bound nucleotide. Under low ionic strength conditions, the approximate dissociation constant (K(D)) for the ADF-actin complex determined from exchange of nucleotide (1,N6-etheno-ATP) is about 150 nM and is calcium-independent. Addition of ADF to monomeric actin inhibits actin assembly as well as the ATP hydrolysis that normally accompanies assembly. Complex formation is demonstrated between ADF and actin containing either ATP, ADP, or AMPPNP as the bound nucleotide. A K(D) of 0.1-0.2 muM was calculated for both the ADF-ATP-actin and ADF-AMPPNP-actin complexes, whereas the K(D) for the ADF-ADP-actin complex is about 1.3 muM. ADF can either depolymerize or cosediment with F-actin in a stoichiometric fashion, but these reciprocal activities are pH-dependent. At pHs between 6.5 and 7.1, ADF cosediments with F-actin and demonstrates only weak depolymerizing activity. ADF binding is cooperative and saturates at a 1:1 ADF:actin molar ratio. At pHs between 7.1 and 7.7, ADF shows increasing depolymerizing activity and less F-actin binding. At pH 8.0, ADF depolymerizes F-actin in a stoichiometric manner. Both the F-actin binding and the depolymerizing activities of ADF are inhibited by phalloidin. At pH 8.0, substoichiometric quantities of ADF, but not ADF-actin complex, transiently enhance the nucleating ability of F-actin solutions in a calcium-insensitive manner, suggesting that ADF has a weak severing activity, but does not cap the filament ends in a manner that inhibits the restoration of the original filament number. We conclude from these results that ADF is a noncapping, calcium-independent, pH sensitive F-actin binding/severing protein which is capable of forming moderate affinity complexes with actin monomers in which the nucleotide exchange is inhibited. The significance of these findings to a presumed cellular role for ADF is discussed.