title: Purification of the neurotensin receptor from mouse brain by affinity chromatography.
abstract: The neurotensin receptor was purified from newborn mouse brain by affinity chromatography. Active neurotensin binding sites were solubilized from brain homogenates using the nondenaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in the presence of cholesteryl hemisuccinate. Chromatography of the soluble extract on SP-Sephadex C-25 and hydroxylapatite eliminated 50% of proteins without loss of neurotensin binding activity. This prepurified material was loaded into an affinity column prepared by coupling neurotensin (2-13) to glutaraldehyde-activated Ultrogel AcA22. Nonspecifically adsorbed proteins were eliminated by extensive washing, and the receptor was eluted with a buffer containing 1 M NaCl, 0.1% CHAPS, and 0.02% cholesteryl hemisuccinate. After desalting, the purified receptor bound 125I-labeled neurotensin with a dissociation constant of 0.26 nM and retained its specificity towards a series of neurotensin analogues. The desalted NaCl eluate appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major band of molecular weight 100,000 which was identified as the receptor by affinity labeling with 125I-labeled neurotensin in the presence of disuccinimidyl suberate. The purity of the mouse brain receptor eluted from the affinity column was estimated to be 78%. Electroelution of the 100-kDa protein band gave an homogenous preparation of receptor. Very similar results were obtained with CHAPS-solubilized neurotensin receptors from rat and rabbit brain.
authors.affiliation: Centre de Biochimie du Centre National de la Recherche Scientifique, Université de Nice, Faculté des Sciences, France.
authors.forename: J
authors.initials: J
authors.lastname: Mazella
authors.forename: J
authors.initials: J
authors.lastname: Chabry
authors.forename: N
authors.initials: N
authors.lastname: Zsurger
authors.forename: J P
authors.initials: JP
authors.lastname: Vincent
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002793:Cholic Acids; D002846:Chromatography, Affinity / Q000379:methods; D003902:Detergents; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009496:Neurotensin / Q000378:metabolism*; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002793:Cholic Acids; D003902:Detergents; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter; D009496:Neurotensin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate