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

A. R.H. Ahmed, G. W.J. Olivier, G. Adams, M. E. Erskine, R. G. Kinsman, S. K. Branch, S. H. Moss, L. J. Notarianni, C. W. Pouton

Research output: Contribution to journalArticleResearchpeer-review

30 Citations (Scopus)

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 languageEnglish
Pages (from-to)377-382
Number of pages6
JournalBiochemical Journal
Volume286
Issue number2
DOIs
Publication statusPublished - 1992
Externally publishedYes

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

Cite this