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.
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.
Ahmed
A R
AR
School of Pharmacy and Pharmacology, University of Bath, U.K.
Olivier
G W
GW
Adams
G
G
Erskine
M E
ME
Kinsman
R G
RG
Branch
S K
SK
Moss
S H
SH
Notarianni
L J
LJ
Pouton
C W
CW
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001426:Bacterial Proteins; D001710:Biotin; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D008280:Magnetics; D008545:Melanoma; D051379:Mice; D008863:Microspheres; D008969:Molecular Sequence Data; D017830:Octoxynol; D010777:Photochemistry; D011092:Polyethylene Glycols; D011979:Receptors, Pituitary Hormone / Q000302:isolation & purification* / Q000378:metabolism; D019809:Streptavidin; D014407:Tumor Cells, Cultured; D000521:alpha-MSH / Q000378:metabolism*
D000345:Affinity Labels; D001426:Bacterial Proteins; D003902:Detergents; D011979:Receptors, Pituitary Hormone; D011092:Polyethylene Glycols; D000521:alpha-MSH; D001710:Biotin; C029446:MSH receptor; D017830:Octoxynol; D019809:Streptavidin