Quirk S, Maciver S K, Ampe C, Doberstein S K, Kaiser D A, VanDamme J, Vandekerckhove J S, Pollard T D
Department of Biophysics and Biophysical and Biophysical Chemistry, Johns Hopkins Medical School, Baltimore, Maryland 21205.
Biochemistry. 1993 Aug 24;32(33):8525-33. doi: 10.1021/bi00084a019.
We determined the amino acid sequence of the actin monomer binding/actin filament severing protein actophorin from Acanthamoeba castellanii by automated Edman degradation of peptide fragments and by sequencing of full-length cDNA. Actophorin consists of 138 amino acids (calculated molecular weight of 15,543) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially vertebrate cofilin, vertebrate actin depolymerizing factor/destrin, and echinoderm depactin. Actophorin is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that actophorin is a single-copy gene; however, Northern blots show two distinct mRNA species of 1 and 0.9 kb in size. Homogeneous recombinant actophorin purified from Escherichia coli is indistinguishable from the native protein in its physical properties and in biochemical assays of its interaction with actin, but is less reactive with three monoclonal antibodies raised against the native protein. The NH2 terminus of native actophrin is blocked, while the initiating methionine residue is removed from recombinant actophorin. This difference has no measurable effect on activity. By fluorescent antibody staining of Acanthamoeba, actophorin colocalizes with actin filaments in the cortical cytoplasm, especially at the leading edge of the cell. Additionally, actophorin binds phosphatidylinositol 4',5'-bisphosphate. The recombinant actophorin forms X-ray diffraction quality crystals of superior quality in poly(ethylene glycol)/2-propanol and, like the native crystal form, belongs to space group P2(1)2(1)2(1).
我们通过对肽片段进行自动埃德曼降解以及对全长cDNA进行测序，确定了来自卡氏棘阿米巴的肌动蛋白单体结合/肌动蛋白丝切断蛋白肌动蛋白结合蛋白的氨基酸序列。肌动蛋白结合蛋白由138个氨基酸组成（计算分子量为15,543），与其他低分子量肌动蛋白单体隔离蛋白具有高度的序列相似性，尤其是脊椎动物的丝切蛋白、脊椎动物的肌动蛋白解聚因子/去整合素，以及棘皮动物的去聚合蛋白。肌动蛋白结合蛋白比相关的脊椎动物蛋白小，且不包含核定位序列。Southern印迹分析表明肌动蛋白结合蛋白是单拷贝基因；然而，Northern印迹显示有两种大小分别为1 kb和0.9 kb的不同mRNA种类。从大肠杆菌中纯化得到的均一重组肌动蛋白结合蛋白，在其物理性质以及与肌动蛋白相互作用的生化分析中与天然蛋白无法区分，但与针对天然蛋白产生的三种单克隆抗体的反应性较低。天然肌动蛋白结合蛋白的NH2末端被封闭，而重组肌动蛋白结合蛋白的起始甲硫氨酸残基被去除。这种差异对活性没有可测量的影响。通过对棘阿米巴进行荧光抗体染色，肌动蛋白结合蛋白与皮质细胞质中的肌动蛋白丝共定位，尤其是在细胞的前缘。此外，肌动蛋白结合蛋白结合磷脂酰肌醇4',5'-二磷酸。重组肌动蛋白结合蛋白在聚（乙二醇）/2-丙醇中形成了高质量的X射线衍射晶体，并且与天然晶体形式一样，属于空间群P2(1)2(1)2(1)。
