Fujita H, Allen P G, Janmey P A, Azuma T, Kwiatkowski D J, Stossel T P, Furu-uchi K, Kuzumaki N
Division of Gene Regulation, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan.
Eur J Biochem. 1997 Sep 15;248(3):834-9. doi: 10.1111/j.1432-1033.1997.00834.x.
Gelsolin is a calcium-activated actin-binding protein with six subdomains. The N-terminal (G1) domain is essential for actin-filament-severing activity while other domains within G2-3 position the protein on the filament side allowing G1 to sever. In order to generate reagents capable of competitively inhibiting endogenous gelsolin and, potentially, other actin filament regulatory protein, we expressed several truncates of gelsolin in Escherichia coli, and analyzed how they affected the in vitro activity of two different actin-binding proteins, gelsolin and cofilin. A Ca2+-sensitive truncate containing G2-6 inhibited the F-actin-depolymerizing activities of both gelsolin and cofilin, while a G2-3 truncate was less effective. Using two independent assays, our results support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kinetics. Cosedimentation assays in the presence of calcium demonstrate that the G2-6 truncate binds to F-actin more strongly than the G2-3 truncate consistent with a protection mechanism by conformational change of F-actin and/or competitive binding to actin filaments which depends upon the presence of actin filament binding domains.
凝溶胶蛋白是一种具有六个亚结构域的钙激活肌动蛋白结合蛋白。N端（G1）结构域对于肌动蛋白丝切断活性至关重要，而G2 - 3内的其他结构域将该蛋白定位在丝的一侧，使G1能够进行切断。为了生成能够竞争性抑制内源性凝溶胶蛋白以及可能的其他肌动蛋白丝调节蛋白的试剂，我们在大肠杆菌中表达了凝溶胶蛋白的几种截短体，并分析了它们如何影响两种不同肌动蛋白结合蛋白（凝溶胶蛋白和丝切蛋白）的体外活性。一种包含G2 - 6的钙敏感截短体抑制了凝溶胶蛋白和丝切蛋白的F - 肌动蛋白解聚活性，而G2 - 3截短体的效果较差。通过两项独立检测，我们的结果支持以下观点：凝溶胶蛋白截短体抑制肌动蛋白丝切断，并且不会显著影响肌动蛋白亚基解离动力学。在有钙存在的情况下进行的共沉降检测表明，G2 - 6截短体比G2 - 3截短体与F - 肌动蛋白的结合更强，这与通过F - 肌动蛋白构象变化的保护机制和/或取决于肌动蛋白丝结合结构域存在的与肌动蛋白丝的竞争性结合一致。
