Actin-bundling protein isolated from pollen tubes of lily. Biochemical and immunocytochemical characterization
Actin-bundling protein isolated from pollen tubes of lily. Biochemical and immunocytochemical characterization
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DOI：
10.1104/pp.116.4.1421
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发表时间：
1998-04
期刊：
影响因子：
7.4
通讯作者：
E. Yokota;Kei-ichiro Takahara;T. Shimmen
中科院分区：
文献类型：
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作者：
E. Yokota;Kei-ichiro Takahara;T. Shimmen
利用与f -肌动蛋白的亲和关系，从百合花粉管中纯化了一个135 kd的肌动蛋白捆绑蛋白。从花粉管的粗提物中，该蛋白与外源添加的f -肌动蛋白共沉淀，然后通过高离子强度溶液处理与f -肌动蛋白分离。通过羟基磷灰石柱、凝胶过滤柱和二乙胺乙基纤维素离子交换柱进一步纯化蛋白质。在本研究中，该蛋白暂定名为P-135-ABP （Plant 135-kD Actin-Bundling protein）。通过凝胶过滤柱的洗脱位置，我们估计纯化的P-135-ABP的天然分子质量为260 kD，表明它在生理条件下以二聚体形式存在。该蛋白以不依赖Ca2+的方式结合并捆绑从鸡胸肌制备的f -肌动蛋白。135-P-ABP与肌动蛋白的结合在26个肌动蛋白单体与1个P-135-ABP二聚体的近似化学计量下饱和。通过透射电镜观察，我们观察到f - actis之间的交叉桥，其纵向周期性为31 nm。使用罗丹明-phalloidin和135-kD多肽抗体的免疫荧光显微镜显示，P-135-ABP与百合花粉管中肌动蛋白细丝束共定位，这使我们得出结论，它是负责细丝束的因素。
A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca2+-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.