The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-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.
|Number of pages||6|
|Volume||286 ( Pt 2)|
|Publication status||Published - 1992|
- 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
Ahmed, A. R., Olivier, G. W., Adams, G., Erskine, M. E., Kinsman, R. G., Branch, S. K., Moss, S. H., Notarianni, L. J., & Pouton, C. W. (1992). Isolation and partial purification of a melanocyte-stimulating hormone receptor from B16 murine melanoma cells. A novel approach using a cleavable biotinylated photoactivated ligand and streptavidin-coated magnetic beads: Biochem J. Biochemical Journal, 286 ( Pt 2), 377-382. http://www.ncbi.nlm.nih.gov/pubmed/1326940