Purification of the neurotensin receptor from mouse brain by affinity chromatography.
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.
Mazella
J
J
Centre de Biochimie du Centre National de la Recherche Scientifique, Université de Nice, Faculté des Sciences, France.
Chabry
J
J
Zsurger
N
N
Vincent
J P
JP
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
D002793:Cholic Acids; D003902:Detergents; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter; D009496:Neurotensin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate