Dufresne M, Poirot S, Jimenez J, Vaysse N, Fourmy D
INSERM U151, CHU Rangueil, Toulouse, France.
Biochimie. 1992 Feb;74(2):149-51. doi: 10.1016/0300-9084(92)90039-h.
So far, no efficient affinity chromatography for CCK receptor purification has been reported that prevented obtention of sequenceable amounts of purified receptor. In this work, 10% of plasma membrane receptor sites were specifically cross-linked with the photoreactive cleavable agonist 125I-ASD-[Thr28, Ahx31]-CCK-25-33, solubilized by NP-40, chromatographied on immobilized wheat germ agglutinin and further immunopurified using anti-CCK antibodies to an overall rate of 3000-3600-fold. Analysis of eluted material demonstrated a protein migrating at Mr 85,000-100,000 and the absence of 35S-labeled impurity. This single and efficient affinity chromatography should provide enough homogeneous receptor protein for microsequence determination and leads to consider immunoaffinity chromatography on immobilized anti-ligand antibodies as a potential tool for purification of membrane receptors.
到目前为止，尚未报道有用于纯化CCK受体的高效亲和色谱法，这使得无法获得可进行测序量的纯化受体。在这项工作中，10%的质膜受体位点与光反应性可裂解激动剂125I-ASD-[Thr28, Ahx31]-CCK-25-33特异性交联，用NP-40溶解，在固定化麦胚凝集素上进行色谱分离，并进一步用抗CCK抗体进行免疫纯化，总体纯化率为3000-3600倍。对洗脱物质的分析表明，有一种蛋白质在Mr 85,000-100,000处迁移，且不存在35S标记的杂质。这种单一且高效的亲和色谱法应为微序列测定提供足够的纯受体蛋白，并促使人们将固定化抗配体抗体上的免疫亲和色谱法视为纯化膜受体的一种潜在工具。
