Actin-Bundling Protein Isolated from Pollen Tubes of Lily

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 Ca(2+)-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.

本研究从百合（Lilium longiflorum）花粉管中，利用其与纤维状肌动蛋白（F-actin）的亲和性，纯化得到一种分子量为135kD的肌动蛋白束集蛋白。从花粉管粗提液中，该蛋白可与外源添加的F-actin共沉淀，随后通过高离子强度溶液处理从F-actin上解离。随后通过羟基磷灰石柱色谱、凝胶过滤柱色谱及二乙氨基乙基纤维素离子交换柱色谱进行连续纯化，进一步获得高纯度的该蛋白。本研究中将该蛋白暂命名为P-135-ABP（Plant 135-kD Actin-Bundling Protein）。通过凝胶过滤柱色谱的洗脱位置，我们估算纯化后的P-135-ABP的天然分子量为260kD，表明其在生理条件下以二聚体形式存在。该蛋白可与从鸡胸肌中制备的F-actin结合并使其束集，且该过程不依赖钙离子（Ca²+）。P-135-ABP与肌动蛋白的结合在约26个肌动蛋白单体对应1个P-135-ABP二聚体的化学计量比时达到饱和。通过透射电子显微镜对超薄切片的观察，我们发现F-actin之间存在纵向周期为31nm的横桥结构。使用罗丹明-鬼笔环肽与抗135kD多肽的抗体进行免疫荧光显微镜观察，结果显示P-135-ABP与百合花粉管内的肌动蛋白丝束共定位，由此我们推断该蛋白正是介导肌动蛋白丝束集的关键因子。