Solubilization and characterization of active neurotensin receptors from mouse brain.
Neurotensin receptors were solubilized from mouse brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). The binding of 125I-labeled [Tyr3]neurotensin to the soluble fraction was time-dependent, saturable, and reversible. Unlabeled neurotensin and its analogues acetylneurotensin (8-13), neurotensin (9-13), and neurotensin (1-12) competitively antagonized the binding of 125I-labeled [Tyr3]neurotensin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane-bound receptors. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of neurotensin binding sites with a Kd of 0.36 nM and a Bm of 63 fmol/mg. As already observed with membrane-bound receptors, the affinity of neurotensin for the soluble binding activity was decreased by Na+ ions. By contrast, soluble receptors were no longer sensitive to GTP and the antihistamine drug levocabastine. A molecular weight of about 100,000 was determined for soluble neurotensin receptors both under native conditions by gel filtration on Ultrogel AcA 34 and under denaturating conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling.
Mazella
J
J
Centre de Biochimie, Université de Nice, Faculté des Sciences, France.
Chabry
J
J
Kitabgi
P
P
Vincent
J P
JP
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002414:Cations, Monovalent; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009496:Neurotensin / Q000378:metabolism*; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D002414:Cations, Monovalent; D002793:Cholic Acids; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter; D009496:Neurotensin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate