FEBS Letters | |
Bombesin receptor from Swiss 3T3 cells Affinity chromatography and reconstitution into phospholipid vesicles | |
Coffer, Arnold1  Sinnett-Smith, James1  Rozengurt, Enrique1  | |
[1] Imperial Cancer Research Fund. PO Box Lincoln's Inn Fields, London WC2A 3PX, UK | |
关键词: Signal transduction; Growth control; Biotinylated bombesin; | |
DOI : 10.1016/0014-5793(90)81462-W | |
学科分类:生物化学/生物物理 | |
来源: John Wiley & Sons Ltd. | |
【 摘 要 】
Bombesin and its mammalian counterpart gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells in which distinct high affinity receptors have been identified. We developed here a probe for specific ligand affinity chromatography by coupling biotin to [lys1]bombesin. The resulting biotinylated [lys3]bombesin (BLB) retained biological activity as judged by inhibition of [125I]GRP binding to intact cells and membrane preparations and stimulation of rapid Ca2+ mobilization and DNA synthesis in intact cells. Using this ligand and magnetised beads coated with streptavidin, we extracted differentially a single protein from detergent-solubilized Swiss 3T3 membranes in a BLB-dependent manner. Visualization was achieved either after autoradiograph of metabolically labelled proteins with [13S]methionine or by silver staining of larger preparations. In other experiments, elution of BLB-receptor complexes bound to streptavidin beads was carried out at neutral pH and eluted fraction was reconstituted into phospholipid vesicles. This procedure revealed[125I]GRP binding activity that exhibited saturability, specificity and a 1946-fold increase in specific activity.
【 授权许可】
Unknown
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