Babich M, Foti L R, Sykaluk L L, Clark C R
Department of Biomedical Sciences, University of Illinois College of Medicine Rockford 61107, USA.
Biochem Biophys Res Commun. 1996 Jan 5;218(1):125-31. doi: 10.1006/bbrc.1996.0022.
Profilin binds to G-actin and affects polymerization. However, regulation of profilin function is generally unknown and controversy exists regarding profilin effects on actin polymerization. Because protein-protein interactions are implicated in many cellular responses, human platelet profilin self-association and actin inter-action was examined. Silver stained SDS-PAGE of poly-l-proline/sepharose 4B column purified profilin revealed the presence of profilin (14.8 kD) and extraneous higher bands (primarily 30 kD and 58.5 kD). Re-electrophoretic analysis of gel electroelution purified profilin yielded predominantly 14.8 kD and 58.5 kD proteins. Rabbit IgG antibodies made against gel electroelution-purified profilin recognized all profilin sizes on immunoblots. Capillary electrophoresis of profilin in solution produced a single peak that resolved into three distinct peaks upon addition of reducing agent or high salt conditions. Further, G-actin did not bind to 14.8 kD profilin on immunoblot overlay assays, but surprisingly bound only to 58.5 kD profilin. The data indicate that monomeric profilin forms tetramers which are the relevant high affinity actin-binding form.
肌动蛋白单体结合蛋白（profilin）与G-肌动蛋白结合并影响其聚合。然而，profilin功能的调节机制通常尚不清楚，并且关于profilin对肌动蛋白聚合的影响存在争议。由于蛋白质-蛋白质相互作用涉及许多细胞反应，因此对人血小板profilin的自缔合和与肌动蛋白的相互作用进行了研究。用聚-L-脯氨酸/琼脂糖4B柱纯化的profilin进行银染SDS-PAGE分析，结果显示存在profilin（14.8 kD）和额外的较高条带（主要为30 kD和58.5 kD）。对凝胶电洗脱纯化的profilin进行再电泳分析，主要得到14.8 kD和58.5 kD的蛋白质。用针对凝胶电洗脱纯化的profilin制备的兔IgG抗体在免疫印迹上识别所有大小的profilin。溶液中profilin的毛细管电泳产生一个单峰，在加入还原剂或高盐条件后分解为三个不同的峰。此外，在免疫印迹覆盖分析中，G-肌动蛋白不与14.8 kD的profilin结合，但令人惊讶的是，它只与58.5 kD的profilin结合。数据表明，单体profilin形成四聚体，这是相关的高亲和力肌动蛋白结合形式。
