Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells
Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells
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DOI：
10.1006/abio.1998.2677
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发表时间：
1998-06-15
影响因子：
2.9
通讯作者：
Broaddus, VC
中科院分区：
文献类型：
--
作者：
Ernst, JD;Yang, L;Broaddus, VC
膜联蛋白特异性结合阴离子磷脂，如磷脂酰丝氨酸，其通常局限于细胞膜的细胞质小叶。在程序性细胞死亡或凋亡期间，这种磷脂不对称性丧失，阴离子磷脂暴露在质膜的细胞外小叶上，在那里它们可接近外源添加的标记的膜联蛋白。化学上[例如，异硫氰酸荧光素（FITC）]修饰的膜联蛋白V已被广泛用于通过流式细胞术检测和计数凋亡细胞。我们制备了含有融合到膜联蛋白V的绿色荧光蛋白（GFP）的嵌合蛋白。含有融合到膜联蛋白V的C-末端的GFP的嵌合体是可溶的和荧光的，但不能结合磷脂。相反，含有GFP融合到膜联蛋白V的N-末端的嵌合体特异性结合凋亡细胞。GFP-膜联蛋白V代表了FITC-annexin V用于细胞凋亡研究的敏感且简便的替代方案。(C)北京：科学出版社.
Annexin proteins specifically bind anionic phospholipids such as phosphatidylserine, which are normally confined to the cytoplasmic leaflet of cellular membranes. During programmed cell death, or apoptosis, this phospholipid asymmetry is lost, and anionic phospholipids are exposed on the extracellular leaflet of the plasma membrane where they are accessible to exogenously added, labeled annexins. Chemically [e.g., fluoroscein isothiocyanate (FITC)]-modified annexin V has been widely used to detect and enumerate apoptotic cells by how cytometry. We prepared chimeric proteins containing green fluorescent protein (GFP) fused to annexin V. A chimera containing GFP fused to the C-terminus of annexin V was soluble and fluorescent, but was unable to bind phospholipids. In contrast, a chimera containing GFP fused to the N-terminus of annexin V specifically bound apoptotic cells. GFP-annexin V represents a sensitive and facile alternative to FITC-annexin V for studies of apoptosis. (C) 1998 Academic Press.