Solubilization and characterization of active neurotensin receptors from mouse brain |
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Authors: | J Mazella J Chabry P Kitabgi J P Vincent |
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Institution: | Centre de Biochimie, Université de Nice, Faculté des Sciences, France. |
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Abstract: | 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. |
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