Kaiser D A, Goldschmidt-Clermont P J, Levine B A, Pollard T D
Department of Cell Biology and Anatomy, Johns Hopkins Medical School, Baltimore, MD 21205.
Cell Motil Cytoskeleton. 1989;14(2):251-62. doi: 10.1002/cm.970140211.
We present evidence that native profilin can be purified from cellular extracts of Acanthamoeba, Dictyostelium, and human platelets by affinity chromatography on poly-L-proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6-8 M. Acanthamoeba profilin-I and profilin-II can subsequently be separated by cation exchange chromatography. The yield of Acanthamoeba profilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: 1) dialyzed Acanthamoeba and human profilins rebind quantitatively to poly-L-proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; 2) dialyzed profilins form 3-D crystals under the same conditions as native profilins; 3) dialyzed Acanthamoeba profilin-I has an NMR spectrum identical with that of native profilin-I; and 4) dialyzed human and Acanthamoeba profilins inhibit actin polymerization. We report the discovery of profilin in Dictyostelium cell extracts using the same method. Based on these observations we conclude that urea elution from poly-L-proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin-II or alpha-actinin in crude cell extracts can be bound selectively to the poly-L-proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin-II from both Acanthamoeba and Dictyostelium cell extracts and alpha-actinin from Acanthamoeba cell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly-L-proline. They can be eluted by dissociating the complexes with ATP and separated from actin by gel filtration if necessary.
我们提供的证据表明，通过在聚-L-脯氨酸琼脂糖上进行亲和层析，可从棘阿米巴、盘基网柄菌和人血小板的细胞提取物中纯化天然的肌动蛋白单体结合蛋白（profilin）。加入细胞提取物并用3M尿素洗涤柱子后，通过将尿素浓度提高到6 - 8M来洗脱均一的肌动蛋白单体结合蛋白。随后，棘阿米巴肌动蛋白单体结合蛋白-I和肌动蛋白单体结合蛋白-II可通过阳离子交换层析分离。棘阿米巴肌动蛋白单体结合蛋白的产量是传统方法的两倍。几条证据表明，通过透析去除尿素后，肌动蛋白单体结合蛋白能完全复性：1）透析后的棘阿米巴和人肌动蛋白单体结合蛋白能定量地重新结合到聚-L-脯氨酸上，并以与未经尿素处理的天然传统纯化肌动蛋白单体结合蛋白相同的方式结合到肌动蛋白上；2）透析后的肌动蛋白单体结合蛋白在与天然肌动蛋白单体结合蛋白相同的条件下形成三维晶体；3）透析后的棘阿米巴肌动蛋白单体结合蛋白-I的核磁共振谱与天然肌动蛋白单体结合蛋白-I的相同；4）透析后的人和棘阿米巴肌动蛋白单体结合蛋白能抑制肌动蛋白聚合。我们报告了使用相同方法在盘基网柄菌细胞提取物中发现肌动蛋白单体结合蛋白。基于这些观察结果，我们得出结论，从聚-L-脯氨酸琼脂糖上用尿素洗脱然后复性，通常将有助于从多种细胞中制备肌动蛋白单体结合蛋白。或许同样具有普遍用途的是这一发现：根据用于平衡柱子的离子条件，粗细胞提取物中的肌球蛋白-II或α-辅肌动蛋白可选择性地结合到聚-L-脯氨酸琼脂糖柱上。我们已在合适的缓冲液中从棘阿米巴和盘基网柄菌细胞提取物中纯化了肌球蛋白-II，并从棘阿米巴细胞提取物中纯化了α-辅肌动蛋白。这些蛋白质通过琼脂糖与肌动蛋白形成复合物而被保留，而非通过与聚-L-脯氨酸的特异性相互作用。如有必要，可通过用ATP解离复合物来洗脱它们，并通过凝胶过滤将它们与肌动蛋白分离。
