Isolation and purification of an Fc epsilon receptor activated ion channel from the rat mast cell line RBL-2H3.-猫眼课题宝

从大鼠肥大细胞系 RBL-2H3 中分离和纯化 Fc ε 受体激活的离子通道。

DOI：

10.1021/bi00419a047

发表时间：

1988

期刊：

Biochemistry

影响因子：

2.9

通讯作者：

中科院分区：

生物学3区

文献类型：

作者：

S. Hemmerich;I. Pecht

合成了抗过敏药克罗塞汀[5，5 '-[（2-羟基-1，3-丙二基）-双（氧基）]双[4-氧代-（4 H-1-苯并吡喃）-2-羧酸]二钠]的衍生物，该衍生物可在丙烷2-位与大分子和不溶性基质共价缀合。检测这些衍生物与大分子的缀合物与大鼠嗜碱性白血病系RBL-2 H3细胞的结合，RBL-2 H3被广泛用作免疫诱导的肥大细胞脱粒的模型。只有那些药物-蛋白质偶联物，其中croplyn类似物与氨基在丙烷2-碳，而不是羟基连接到载体的戊二醛被发现表现出特异性和饱和结合这些细胞。对这些结合物的结合数据进行分析，得出表观结合常数为3.8 +/- 0.2 × 10（8）M-1，探针的表观结合位点数为4000-8000个/细胞。发现特异性结合细胞的缀合物也被固定在琼脂糖基质上，并用于对负责观察到的结合的膜组分进行基于亲和力的分离。从这些柱上洗脱单个标记的多肽，在这些柱上吸附了全细胞裂解物或表面放射性碘标记的RBL-2 H3细胞的溶解的纯化质膜。这种膜蛋白在非还原SDS-PAGE的放射自显影图上显示为表观分子量约为110，000道尔顿（Da）的单一宽带。在还原凝胶的放射自显影图上，检测到的唯一条带具有约50，000 Da的表观质量，并且看起来更窄。用药物和二硫化物还原剂或单独用后者洗脱柱导致50-kDa多肽的产率显著更高。完整的和减少的蛋白质强烈结合到固定化伴刀豆球蛋白A和固定化麦胚凝集素，这表明分离的完整蛋白质可能是一个类似的分子量的两个糖基化亚基的二聚体。用糖苷内切酶F处理还原的蛋白质导致其表观分子量降低约12 kDa，表明该多肽的糖基化程度约为25%。如以下论文所示，完整蛋白质构成在IgE-Fc γ受体聚集时被激活的Ca 2+通道。

Derivatives of the antiallergic drug cromolyn [disodium 5,5'-[(2-hydroxy-1,3-propanediyl)-bis(oxy)]bis [4-oxo-(4H-1-benzopyran)-2- carboxylate]], which can be conjugated covalently at the propane 2-position to macromolecules and to insoluble matrices, were synthesized. Conjugates of these derivatives with macromolecules were examined for their binding to cells of the rat basophilic leukemia line RBL-2H3, which is widely employed as a model for immunologically induced mast cell degranulation. Only those drug-protein conjugates in which the cromolyn analogue with an amino group at the propane 2-carbon instead of the hydroxyl was linked to the carrier by glutaraldehyde were found to exhibit specific and saturable binding to these cells. Analysis of the binding data for these conjugates yielded an apparent binding constant of 3.8 +/- 0.2 X 10(8) M-1 and an apparent number of binding sites for the probe of 4000-8000 per cell. The conjugates found to bind specifically to the cells were also immobilized on agarose matrices and employed in an affinity-based isolation of the membrane component responsible for the observed binding. A single labeled polypeptide was eluted from these columns, onto which either whole cell lysates or solubilized purified plasma membranes of surface-radioiodinated RBL-2H3 cells had been adsorbed. This membrane protein appears on autoradiograms of nonreducing SDS-PAGE as a single broad band of approximately 110,000 daltons (Da) apparent molecular mass. On autoradiograms of reducing gels, the only band detected has an apparent mass of approximately 50,000 Da and appears narrower. Elution of the columns with the drug and disulfide-reducing agents or with the latter alone resulted in significantly higher yields of the 50-kDa polypeptide. Both the intact and reduced proteins bind strongly to immobilized concanavalin A and less so to immobilized wheat germ agglutinin, suggesting that the isolated intact protein is probably a dimer of two glycosylated subunits of similar molecular mass. Treatment of the reduced protein with endoglycosidase F leads to a decrease in its apparent molecular mass by approximately 12 kDa, suggesting that the extent of glycosylation of this polypeptide is approximately 25%. As shown in the following paper, the intact protein constitutes a Ca2+ channel that is activated upon IgE-Fc epsilon receptor aggregation.