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

被引：28

作者：

AHMED, ARH ^{[1
]}

OLIVIER, GWJ ^{[1
]}

ADAMS, G ^{[1
]}

ERSKINE, ME ^{[1
]}

KINSMAN, RG ^{[1
]}

BRANCH, SK ^{[1
]}

MOSS, SH ^{[1
]}

NOTARIANNI, LJ ^{[1
]}

POUTON, CW ^{[1
]}

机构：

[1] UNIV BATH,SCH CHEM,BATH BA2 7AY,AVON,ENGLAND

来源：

BIOCHEMICAL JOURNAL | 1992年 / 286卷

关键词：

D O I：

10.1042/bj2860377

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 streptavidin/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.

引用

页码：377 / 382

页数：6

共 15 条

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