可溶性全长小鼠促红细胞生成素受体的配体结合动力学

Ligand binding kinetics of a soluble full-length murine erythropoietin receptor.

作者信息

Avedissian L S, Poola I, Spivak J L

机构信息

Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

出版信息

Biochem Biophys Res Commun. 1995 Nov 2;216(1):62-8. doi: 10.1006/bbrc.1995.2592.

DOI:10.1006/bbrc.1995.2592

PMID:7488125

Abstract

The full-length murine erythropoietin receptor was expressed in Sf9 cells using a baculovirus vector. Erythropoietin receptors in solubilized Sf9 cell lysates bound erythropoietin with high affinity (92 pM). Erythropoietin receptor-125I-labeled erythropoietin association and dissociation kinetics using solubilized Sf9 cell lysates revealed a ka of 0.16 nM-1 min-1 and a kd of 0.00055 min-1 giving an observed KD of 3.45 pM. The erythropoietin receptors was partially purified from Sf9 cell lysates by chromatography on Con A Sepharose. When erythropoietin receptors were crosslinked to 125I-labeled erythropoietin and analyzed by SDS-7.5% PAGE protein complexes of 90 and 125 kDa were observed with receptors in solubilized lysate, and 170 and 190 kDa with the partially purified receptors.

摘要

使用杆状病毒载体在Sf9细胞中表达全长小鼠促红细胞生成素受体。溶解的Sf9细胞裂解物中的促红细胞生成素受体以高亲和力（92 pM）结合促红细胞生成素。使用溶解的Sf9细胞裂解物进行促红细胞生成素受体 - 125I标记的促红细胞生成素结合和解离动力学分析，得出结合常数（ka）为0.16 nM-1 min-1，解离常数（kd）为0.00055 min-1，观察到的解离常数（KD）为3.45 pM。通过Con A琼脂糖柱层析从Sf9细胞裂解物中部分纯化促红细胞生成素受体。当促红细胞生成素受体与125I标记的促红细胞生成素交联并通过SDS - 7.5% PAGE分析时，在溶解的裂解物中的受体观察到90和125 kDa的蛋白复合物，在部分纯化的受体中观察到170和190 kDa的蛋白复合物。