Larsson H, Lindberg U
Department of Zoological Cell Biology, Wenner-Gren Institute, University of Stockholm, Sweden.
Biochim Biophys Acta. 1988 Mar 2;953(1):95-105. doi: 10.1016/0167-4838(88)90013-1.
The interaction between calf spleen profilin and actin depends critically on the status of the C-terminus of the actin, and in the case of profilin, the C-terminus is of great importance for the physiochemical behaviour of the protein. Both proteins easily lose their C-terminal amino acids during the preparation, and special care has to be taken to ensure the isolation of the proteins in the intact form. Another factor that may seriously influence the study of the interaction of profilin with actin is the presence of varying amounts of an activity that causes an apparent stabilization of the complex even at later stages of its purification. We have found conditions for the isolation of intact profilin and actin, and studied the interaction between the two proteins, including the determination of the Kdiss for the complex formed under various ionic conditions. The complex formed between profilin and actin from calf spleen was found to be significantly stronger (Kdiss less than or equal to 10(-8) M in 50 mM KCl, and Kdiss = 4.10(-7) M in 50 mM KCl, 1 mM MgCl2) than that formed between profilin and muscle alpha-actin (Kdiss = 10(-6) M in 50 mM KCl, +/- 1 mM MgCl2). The profilactin complex formed in the mammalian system was stronger than the complex formed between Acanthamoeba actin and the profilin-like protein isolated from this organism. Analysis of the formation of the calf spleen complex in the presence of varying concentrations of divalent cations gave evidence for the presence of a high-affinity divalent-cation-binding site on the spleen actin (beta, gamma) which appears to regulate the interaction with profilin.
小牛脾肌动蛋白单体结合蛋白与肌动蛋白之间的相互作用关键取决于肌动蛋白C末端的状态，就肌动蛋白单体结合蛋白而言，C末端对该蛋白质的物理化学行为非常重要。在制备过程中，这两种蛋白质都很容易失去其C末端氨基酸，因此必须格外小心以确保完整形式的蛋白质的分离。另一个可能严重影响肌动蛋白单体结合蛋白与肌动蛋白相互作用研究的因素是存在不同量的一种活性物质，即使在其纯化的后期阶段，这种活性物质也会导致复合物明显稳定。我们已经找到了完整分离肌动蛋白单体结合蛋白和肌动蛋白的条件，并研究了这两种蛋白质之间的相互作用，包括测定在各种离子条件下形成的复合物的解离常数（Kdiss）。发现小牛脾中的肌动蛋白单体结合蛋白与肌动蛋白形成的复合物比肌动蛋白单体结合蛋白与肌肉α-肌动蛋白形成的复合物（在50 mM KCl，±1 mM MgCl2中Kdiss = 10(-6) M）要强得多（在50 mM KCl中Kdiss小于或等于10(-8) M，在50 mM KCl，1 mM MgCl2中Kdiss = 4.10(-7) M）。在哺乳动物系统中形成的肌动蛋白单体结合蛋白 - 肌动蛋白复合物比棘阿米巴肌动蛋白与从该生物体中分离出的肌动蛋白单体结合蛋白样蛋白质之间形成的复合物更强。在不同浓度二价阳离子存在下对小牛脾复合物形成的分析表明，脾肌动蛋白（β，γ）上存在一个高亲和力的二价阳离子结合位点，该位点似乎调节与肌动蛋白单体结合蛋白的相互作用。
