Giehl K, Valenta R, Rothkegel M, Ronsiek M, Mannherz H G, Jockusch B M
University of Bielefeld, Germany.
Eur J Biochem. 1994 Dec 1;226(2):681-9. doi: 10.1111/j.1432-1033.1994.tb20096.x.
The mode of interaction of birch and bovine profilins with actin was compared using a number of techniques. Birch profilin was purified from pollen or as a recombinant protein from Escherichia coli, using poly(L-proline) affinity chromatography and a monoclonal antibody for the identification of the isolated product. On two-dimensional gels, the genuine and recombinant proteins were identical in molecular mass and isoelectric point and revealed that birch profilin, in contrast to the basic profilins found in mammals, is an acidic protein, analogous to maize profilins. Bovine profilin was obtained from calf thymus. In viscometric assays, the birch protein was seen to modulate actin filament formation analogous to animal profilin. Birch profilin increased the critical concentration required for muscle and brain actin polymerization in a concentration-dependent manner, supporting the notion of the formation of a heterologous complex between the plant protein and animal actin. The effect was Mg(2+)-sensitive, as had been described for homologous complexes. The dissociation constants obtained for the plant/vertebrate and the vertebrate/vertebrate system were both in the micromolar range. The affinity of birch profilin for muscle actin was slightly lower than that for nonmuscle (brain) actin. A binary complex of birch profilin and skeletal muscle actin could be isolated by gel chromatography. Cross-linking experiments with actin, birch profilin, the G-actin binding peptide thymosin beta 4 and gelsolin segment 1, the N-terminal fragment of an actin capping protein, showed that profilin competed with thymosin beta 4, but had no effect on segment 1 binding to actin. These data indicate that the actin-binding domains in plant and animal profilins are functionally highly conserved, although the overall sequence similarity is less than 25%.
运用多种技术比较了桦树肌动蛋白结合蛋白和牛肌动蛋白结合蛋白与肌动蛋白的相互作用模式。桦树肌动蛋白结合蛋白从花粉中纯化得到，或通过大肠杆菌重组蛋白获得，采用聚（L-脯氨酸）亲和色谱法和单克隆抗体鉴定分离产物。在二维凝胶上，天然蛋白和重组蛋白的分子量和等电点相同，表明桦树肌动蛋白结合蛋白与哺乳动物中发现的碱性肌动蛋白结合蛋白不同，是一种酸性蛋白，类似于玉米肌动蛋白结合蛋白。牛肌动蛋白结合蛋白从小牛胸腺中获得。在粘度测定实验中，发现桦树蛋白与动物肌动蛋白结合蛋白类似，能够调节肌动蛋白丝的形成。桦树肌动蛋白结合蛋白以浓度依赖的方式增加了肌肉和脑肌动蛋白聚合所需的临界浓度，支持了植物蛋白和动物肌动蛋白之间形成异源复合物的观点。正如同源复合物所描述的那样，这种效应对Mg(2+)敏感。植物/脊椎动物系统和脊椎动物/脊椎动物系统的解离常数均在微摩尔范围内。桦树肌动蛋白结合蛋白对肌肉肌动蛋白的亲和力略低于对非肌肉（脑）肌动蛋白的亲和力。通过凝胶色谱法可以分离出桦树肌动蛋白结合蛋白和骨骼肌肌动蛋白的二元复合物。用肌动蛋白、桦树肌动蛋白结合蛋白、G-肌动蛋白结合肽胸腺素β4和凝溶胶蛋白片段1（一种肌动蛋白封端蛋白的N端片段）进行交联实验，结果表明肌动蛋白结合蛋白与胸腺素β4竞争，但对片段1与肌动蛋白的结合没有影响。这些数据表明，尽管植物和动物肌动蛋白结合蛋白的整体序列相似性小于25%，但它们的肌动蛋白结合结构域在功能上高度保守。
