Bock D, Hinssen H, D'Haese J
Institut für Zoomorphologie und Zellbiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Eur J Biochem. 1994 Oct 15;225(2):727-35. doi: 10.1111/j.1432-1033.1994.00727.x.
A Ca(2+)-dependent actin-severing protein was purified from the tail muscle of the crayfish Astacus leptodactylus. The isolation procedure involved extraction at low ionic strength in the presence of EGTA, followed by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The purified crayfish actin modulator appeared as a single band with a molecular mass of 105 kDa on SDS/PAGE. The crustacean actin modulator revealed basic functional properties in common with vertebrate gelsolin, like the Ca(2+)-activated severing of F-actin and the nucleation of actin polymerization. However, both proteins differed in major aspects: Ca2+ activation of crayfish actin modulator started at lower threshold concentrations (0.1 microM). The effect of the modulator on shortening the nucleation phase of actin polymerization was significantly weaker at lower modulator/actin ratios. The modulator formed three distinct stoichiometric complexes with G-actin, identified as binary, ternary and quaternary. Binding of G-actin occurred in a low cooperative manner and was completely reversible by EGTA. Despite some properties being similar to those of villin, crayfish actin modulator did not cross-link actin filaments. It is regarded in principle as a gelsolin-type protein, but with characteristic functional deviations from vertebrate gelsolin.
从小龙虾细足螯虾的尾部肌肉中纯化出一种钙依赖性肌动蛋白切断蛋白。分离过程包括在EGTA存在下于低离子强度下提取，随后进行硫酸铵分级分离、离子交换色谱和凝胶过滤。纯化后的小龙虾肌动蛋白调节剂在SDS/PAGE上呈现为一条分子量为105 kDa的单带。这种甲壳类动物肌动蛋白调节剂显示出与脊椎动物凝溶胶蛋白共有的基本功能特性，如F-肌动蛋白的钙激活切断和肌动蛋白聚合的成核作用。然而，这两种蛋白质在主要方面存在差异：小龙虾肌动蛋白调节剂的钙激活始于较低的阈值浓度（0.1 microM）。在较低的调节剂/肌动蛋白比率下，调节剂对缩短肌动蛋白聚合成核阶段的作用明显较弱。该调节剂与G-肌动蛋白形成三种不同的化学计量复合物，分别鉴定为二元、三元和四元复合物。G-肌动蛋白的结合以低协同方式发生，并且可被EGTA完全逆转。尽管小龙虾肌动蛋白调节剂的一些特性与绒毛蛋白相似，但它不会交联肌动蛋白丝。原则上它被视为一种凝溶胶蛋白型蛋白质，但与脊椎动物凝溶胶蛋白存在功能性差异。
