Moroianu J, Fett J W, Riordan J F, Vallee B L
Center for Biochemical and Biophysical Science and Medicine, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1993 May 1;90(9):3815-9. doi: 10.1073/pnas.90.9.3815.
An angiogenin binding protein isolated previously from endothelial cells has been shown to be a member of the actin family. Calf pulmonary artery endothelial (CPAE) cells were investigated for the presence of surface actin by immunoblotting of isolated surface proteins and by immunofluorescence. CPAE cell surface proteins were isolated by selective apical biotinylation and recovery of biotinylated proteins by avidin affinity chromatography. Immunoblotting with a specific smooth muscle alpha-actin antibody detected the presence of this type of actin among the isolated cell surface proteins. Immunofluorescence confirmed that smooth muscle alpha-actin is localized at the surface of nonpermeabilized CPAE cells. Exposure of CPAE cells to angiogenin prior to cell surface immunostaining diminished the signal. When CPAE and rat aortic smooth muscle cells were made permeable before staining, stress fibers could be recognized by the antibody in smooth muscle cells but not CPAE cells. The results indicate that a smooth muscle type of alpha-actin is localized specifically on the surface of cultured CPAE cells where it might interact with angiogenin and other actin binding proteins present in the extracellular environment.
先前从内皮细胞中分离出的一种血管生成素结合蛋白已被证明是肌动蛋白家族的一员。通过对分离出的表面蛋白进行免疫印迹和免疫荧光,研究了小牛肺动脉内皮(CPAE)细胞表面肌动蛋白的存在情况。通过选择性顶端生物素化分离CPAE细胞表面蛋白,并通过抗生物素蛋白亲和色谱法回收生物素化蛋白。用特异性平滑肌α-肌动蛋白抗体进行免疫印迹,在分离出的细胞表面蛋白中检测到了这种类型的肌动蛋白。免疫荧光证实平滑肌α-肌动蛋白定位于未通透的CPAE细胞表面。在细胞表面免疫染色之前,将CPAE细胞暴露于血管生成素会减弱信号。当在染色前使CPAE细胞和大鼠主动脉平滑肌细胞通透时,平滑肌细胞中的抗体可识别应力纤维,而CPAE细胞中则不能。结果表明,平滑肌类型的α-肌动蛋白特异性定位于培养的CPAE细胞表面,在那里它可能与细胞外环境中存在的血管生成素和其他肌动蛋白结合蛋白相互作用。