Abstract
The α-melanocyte-stimulating hormone (α-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of α-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptavidin/biotin-based affinity system was used to isolate the α-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled α-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the α-MSH receptor. Streptavidin-coated magnetic beads were used as a solid support instead of an affinity column. Covalently linked probe-receptor complexes solubilized in Triton X-100 were equilibrated with the beads, and after magnetic separation and washing, specifically bound complexes were treated with dithiothreitol to cleave the disulphide bridge in the biotin-peptide spacer arm and so release the receptor-ligand complex. The identity of the isolated protein was established by SDS/PAGE analysis. Methods to achieve purification to homogeneity and to allow quantitative isolation of the receptor are discussed.
Original language | English |
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Pages (from-to) | 377-382 |
Number of pages | 6 |
Journal | Biochemical Journal |
Volume | 286 |
Issue number | 2 |
DOIs | |
Publication status | Published - 1992 |
Externally published | Yes |
Keywords
- Affinity Labels Amino Acid Sequence Animals Autoradiography *Bacterial Proteins *Biotin Detergents Electrophoresis, Polyacrylamide Gel Magnetics Melanoma Mice Microspheres Molecular Sequence Data Octoxynol Photochemistry Polyethylene Glycols Receptors, Pituitary Hormone/*isolation & purification/metabolism Streptavidin Tumor Cells, Cultured alpha-MSH/*metabolism