Detection and isolation of the erythropoietin receptor using biotinylated erythropoietin.
Detection and isolation of the erythropoietin receptor using biotinylated erythropoietin.
复制标题
使用生物素化促红细胞生成素检测和分离促红细胞生成素受体。
作者：
A. W. Wognum;Peter M. Lansdorp;R. Humphries;Gerald Krystal
已经开发出用生物素标记人促红细胞生成素 (Ep) 的程序，以检测和分离 Ep 受体。该标记方法利用 Ep 上丰富的碳水化合物基团，得到具有生物活性的生物素-Ep (b-Ep)，每个 Ep 分子含有 8 至 10 个生物素。使用 125I 标记的链霉亲和素证明了 b-Ep 与因注射苯肼而贫血的小鼠脾脏细胞的特异性结合。研究发现，B-Ep 与荧光标记的链霉亲和素一起可以通过流式细胞术特异性检测携带 Ep 受体的细胞。这通过多种方式得到了证明。首先，用 b-Ep 和链霉亲和素-藻红蛋白染色后，苯肼处理小鼠的有核脾细胞中约 90% 发出清晰的荧光，而未处理小鼠的脾细胞仅检测到背景荧光。此外，在 5% 至 10% 的正常小鼠骨髓细胞上检测到 Ep 受体，并且通过根据光散射特性将细胞分成亚群，可以将这些细胞本质上鉴定为红系细胞。第三，发现 Ep 受体表达与转铁蛋白受体表达呈正相关，证实了这些细胞的红系性质。 B-Ep 还用于从用鼠 Ep 受体 cDNA 转染的猴 COS 细胞中分离 Ep 受体。在这些实验中，从这些细胞中分离出约65Kd的细胞表面结合蛋白和约60Kd的细胞内蛋白。本报告中描述的检测 Ep 受体表达细胞和分离 Ep 受体的程序对于从异质细胞群中纯化红系细胞、阐明 Ep 受体的结构以及在细胞和分子水平上研究 Ep 的生物活性具有重要意义。
Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin. B-Ep, together with fluorescently tagged streptavidin, was found to specifically detect Ep-receptor-bearing cells by flow cytometry. This was demonstrated in several ways. First, approximately 90% of nucleated spleen cells from phenylhydrazine-treated mice were clearly fluorescent after staining with b-Ep and streptavidin-phycoerythrin, whereas only background fluorescence was detected using spleen cells from untreated mice. In addition, Ep-receptors were detected on 5% to 10% of normal mouse bone marrow cells, and these cells could be identified as erythroid in nature by separating the cells into subpopulations based on light-scatter properties. Third, Ep-receptor expression was found to correlate positively with expression of transferrin receptors, confirming the erythroid nature of these cells. B-Ep was also used to isolate Ep-receptors from monkey COS cells transfected with the murine Ep-receptor cDNA. In these experiments a cell-surface-bound protein of approximately 65 Kd and an intracellular protein of approximately 60 Kd were isolated from these cells. The procedures described in this report for detecting Ep-receptor expressing cells and for isolating the Ep-receptor should be valuable for purifying erythroid cells from heterogeneous cell populations, for elucidating the structure of the Ep-receptor, and for studying the biological activities of Ep at the cellular and molecular level.
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DOI：
10.1016/s0021-9258(18)45317-3
发表时间：
1987-09
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hiroshi Sasakis;B. Bothner;A. Dell;Minoru Fukudall
通讯作者：
Hiroshi Sasakis;B. Bothner;A. Dell;Minoru Fukudall
生物素化重组人促红细胞生成素：生物活性和作为受体配体的用途。
影响因子：
20.3
作者：
Wojchowski,DM;Caslake,L
通讯作者：
Caslake,L
影响因子：
20.3
作者：
M. Loken;V. O. Shah;K. Dattilio;C. Civin
通讯作者：
M. Loken;V. O. Shah;K. Dattilio;C. Civin
通过与弗兰德病毒感染的红细胞交联来鉴定促红细胞生成素受体。
DOI：
10.1073/pnas.84.11.3690
发表时间：
1987
影响因子：
11.1
作者：
Sawyer,ST;Krantz,SB;Luna,J
通讯作者：
Luna,J
弗兰德病毒感染的红细胞中促红细胞生成素的结合和受体介导的内吞作用。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Sawyer,ST;Krantz,SB;Goldwasser,E
通讯作者：
Goldwasser,E