Lheureux K, Forné T, Chaussepied P
Centre de Recherches de Biochimie Macromoléculaire, CNRS-UPR9008, INSERM-U249, Montpellier, France.
Biochemistry. 1993 Sep 28;32(38):10005-14. doi: 10.1021/bi00089a016.
The properties of polymerization and interaction of the G-actin-myosin S1 complexes (formed with either the S1(A1) or the S1(A2) isoform) have been studied by light-scattering and fluorescence measurements in the absence and in the presence of DNase I. In the absence of DNase I, the G-actin-S1(A1) and G-actin-S1(A2) complexes were found to be characterized by different limiting concentrations (l.c.), defined as the complex concentrations above which the polymerization occurs spontaneously within 20 h at 20 degrees C in a "no salt" buffer (l.c. = 0.42 and 8.8 microM for G-actin-S1(A1) and G-actin-S1(A2), respectively). The occurrence of a limiting concentration for either complex together with the kinetic properties of the polymerization led us to conclude that the G-actin-S1 polymerization occurs via a nucleation-elongation process. Fluorescence titrations and proteolysis experiments revealed that G-actin interacts with S1 with a 1:1 stoichiometry (independently of the presence of ATP) with dissociation constants, in the absence of nucleotide, of 20 and 50 nM for the G-actin-S1(A1) and G-actin-S1(A2) complexes, respectively. In the presence of at least a 1.5-fold excess of DNase I, the polymerization of the G-actin-S1 complexes was blocked even at high protein concentration or in the presence of salts. In addition, the affinity of either S1 isoform to actin was reduced 4-5-fold by DNase I, while the stoichiometry of the G-actin-S1 complexes was not changed. However, since the dissociation constants remain in the submicromolar range, we could demonstrate the existence of ternary DNase I-G-actin-S1 complexes stable under polymerizing conditions. Finally, the study of the effect of nucleotides and of various salts on the G-actin-S1 interaction further showed significant differences between the G-actin-S1 and F-actin-S1 interactions.
通过光散射和荧光测量，在不存在和存在脱氧核糖核酸酶I（DNase I）的情况下，研究了G-肌动蛋白-肌球蛋白S1复合物（由S1(A1)或S1(A2)亚型形成）的聚合特性和相互作用。在不存在DNase I的情况下，发现G-肌动蛋白-S1(A1)和G-肌动蛋白-S1(A2)复合物具有不同的极限浓度（l.c.），其定义为在“无盐”缓冲液中于20℃下20小时内自发发生聚合的复合物浓度（G-肌动蛋白-S1(A1)和G-肌动蛋白-S1(A2)的l.c.分别为0.42和8.8 microM）。任一复合物极限浓度的出现以及聚合的动力学特性使我们得出结论，G-肌动蛋白-S1聚合通过成核-延伸过程发生。荧光滴定和蛋白水解实验表明，G-肌动蛋白与S1以1:1的化学计量比相互作用（与ATP的存在无关），在不存在核苷酸的情况下，G-肌动蛋白-S1(A1)和G-肌动蛋白-S1(A2)复合物的解离常数分别为20和50 nM。在至少存在1.5倍过量DNase I的情况下，即使在高蛋白浓度或存在盐的情况下，G-肌动蛋白-S1复合物的聚合也会被阻断。此外，DNase I使任一S1亚型与肌动蛋白的亲和力降低4至5倍，而G-肌动蛋白-S1复合物的化学计量比不变。然而，由于解离常数仍处于亚微摩尔范围内，我们能够证明在聚合条件下稳定存在三元DNase I-G-肌动蛋白-S1复合物。最后，对核苷酸和各种盐对G-肌动蛋白-S1相互作用影响的研究进一步表明，G-肌动蛋白-S1和F-肌动蛋白-S1相互作用之间存在显著差异。
