Strzelecka-Gołaszewska H, Mossakowska M, Woźniak A, Moraczewska J, Nakayama H
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Biochem J. 1995 Apr 15;307 ( Pt 2)(Pt 2):527-34. doi: 10.1042/bj3070527.
Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process.
肌动蛋白的截短衍生物，即缺失最后两个残基（肌动蛋白 - 2C）或三个残基（肌动蛋白 - 3C）的衍生物，被用于研究C末端片段在肌动蛋白聚合中的作用。单体临界浓度和聚合速率按以下顺序增加：完整肌动蛋白＜肌动蛋白 - 2C＜肌动蛋白 - 3C。相反，在Mg - 肌动蛋白自发聚合过程中，肌动蛋白结合的ATP水解速率按相同顺序降低，因此，对于肌动蛋白 - 3C，ATP水解明显滞后于聚合物生长。通过测量结合核苷酸交换速率来探测单体形式下核苷酸位点的构象，结果显示从C末端去除两个或三个残基时会出现类似变化。C末端截短还导致单体肌动蛋白在DNA酶I结合环内的枯草杆菌蛋白酶切割速率略有下降，而在F - 肌动蛋白亚基中，该环内此位点以及另一个被大肠杆菌A2菌株蛋白酶特异性切割的位点的敏感性，随着从C末端依次去除两个和第三个残基而逐渐增加。从这项工作中的这些以及其他观察结果可以得出结论，单体肌动蛋白C末端结构的扰动会传递到结合核苷酸和二价阳离子的裂隙以及亚结构域2顶部的DNA酶I结合环。在聚合物水平上观察到的这些位点的进一步变化，似乎是由于C末端残基与DNA酶I结合环之间的亚基间接触被消除所致。有人提出，这种接触的形成在调节与聚合过程相关的肌动蛋白结合的ATP水解中起着至关重要的作用。
