Kasprzak A A
Centre de Recherches de Biochimie Macromoléculaire, Université de Montpellier I, France.
J Biol Chem. 1993 Jun 25;268(18):13261-6.
The dissociation rates of 1,N6-ethenoadenosine 5'-triphosphate (epsilon ATP) and of Ca2+ from G-actin and its complex with myosin subfragment 1 (S1) were measured by recording a large decrease in the fluorescence intensity of the dissociating nucleotide. Under the experimental conditions employed, the binary G-acto-S1A2 complex does not polymerize (Chaussepied, P., and Kasprzak, A. A. (1989) Nature 342, 950-953). The released nucleotide was hydrolyzed either by alkaline phosphatase or by apyrase; to trap Ca2+, EDTA was used. From the anisotropy of N-iodoacetyl-N'-(5-sulfo-1- naphthyl)ethylenediamine (1,5-IAEDANS)-actin, it was established that during the dissociation of epsilon ATP, the G-acto-S1 complex remained stable and the equilibrium of the system was unaltered. The reactions followed first order kinetics. The dissociation rate constant, kd for epsilon ATP decreased from 5.5 x 10(-4) s-1 for free G-actin to 1 x 10(-4) s-1 for G-acto-S1A2; for Ca2+, kd was also similarly reduced from 2.8 x 10(-2) s-1 to 4 x 10(-3) s-1. Two proteolytically derived actin variants were also examined. For free subtilisin-cleaved actin, kd for epsilon ATP was elevated 2-fold but was almost unchanged for Ca2+. In the complex of the cleaved G-actin with S1A2, kd for both epsilon ATP and for Ca2+ were reduced. The removal of the last 3 amino acids from actin produced a derivative whose behavior in binding to S1, as well as in the kinetics of epsilon ATP and Ca2+ dissociation, was undistinguishable from the unmodified protein.
通过记录解离核苷酸荧光强度的大幅下降，测定了1,N6 - 乙烯腺苷5'-三磷酸（ε-ATP）和Ca2+从G - 肌动蛋白及其与肌球蛋白亚片段1（S1）的复合物中的解离速率。在所采用的实验条件下，二元G - acto - S1A2复合物不会聚合（Chaussepied, P., 和Kasprzak, A. A. (1989) Nature 342, 950 - 953）。释放的核苷酸可通过碱性磷酸酶或腺苷三磷酸双磷酸酶水解；为捕获Ca2+，使用了乙二胺四乙酸（EDTA）。根据N - 碘乙酰 - N' -（5 - 磺基 - 1 - 萘基）乙二胺（1,5 - IAEDANS） - 肌动蛋白的各向异性，确定在ε-ATP解离过程中，G - acto - S1复合物保持稳定，系统平衡未改变。反应遵循一级动力学。ε-ATP的解离速率常数kd从游离G - 肌动蛋白的5.5×10(-4) s-1降至G - acto - S1A2的1×10(-4) s-1；对于Ca2+，kd也类似地从2.8×10(-2) s-1降至4×10(-3) s-1。还研究了两种经蛋白酶水解得到的肌动蛋白变体。对于游离的枯草杆菌蛋白酶切割的肌动蛋白，ε-ATP的kd升高了2倍，但Ca2+的kd几乎不变。在切割的G - 肌动蛋白与S1A2的复合物中，ε-ATP和Ca2+的kd均降低。从肌动蛋白中去除最后3个氨基酸产生了一种衍生物，其与S1结合的行为以及ε-ATP和Ca2+解离的动力学与未修饰的蛋白质无法区分。
