Shojaee N, Patton W F, Chung-Welch N, Su Q, Hechtman H B, Shepro D
Microvascular Research Laboratory, Biological Science Center, Boston University, MA, USA.
Electrophoresis. 1998 Feb;19(2):323-32. doi: 10.1002/elps.1150190230.
Two principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN-1 and FLN-2). A protein that copurifies with an alpha-naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed-phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60-66% identity with nonmuscle filamin (ABP-280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16-residue synthetic peptide corresponding to a structural beta-sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non-muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical vein EC and at least three times the amount expressed in human omentum microvessel and bovine pulmonary artery EC. Differential detergent fractionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, alpha-actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.
非肌肉型和肌肉型同种型（FLN - 1和FLN - 2）。通过非变性电泳、十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳和电印迹法，从人网膜微血管内皮细胞（EC）中分离出一种与α-萘乙酸水解酯酶共纯化的蛋白质。将纯化后的蛋白质进行原位胰蛋白酶切割、反相高效液相色谱（HPLC）和自动Edman降解。鉴定出该蛋白质的六个肽片段与非肌肉细丝蛋白（ABP - 280）有60 - 66%的同源性。其中两个肽段与先前测序的人肌肉细丝蛋白片段100%相同。使用对应于肌肉细丝蛋白结构β折叠区域的16个残基合成肽产生多克隆抗体。与所评估的多种血管细胞相比，视网膜周细胞表达大量的肌肉型和非肌肉型细丝蛋白同种型。周细胞中肌肉细丝蛋白的含量至少是人脐静脉内皮细胞的10倍，是人网膜微血管和牛肺动脉内皮细胞中表达量的至少3倍。差异去污剂分级分离表明，两种细丝蛋白同种型主要定位于周细胞的细胞质和膜/细胞器部分。另一种肌动蛋白交联蛋白α辅肌动蛋白主要存在于细胞质和细胞骨架部分。周细胞中肌动蛋白微丝组织的动态调节可能部分受两种细丝蛋白同种型的控制，而这反过来可能有助于周细胞的收缩性。
