Chromatographic analyses of the serotonin 5-HT1A receptor solubilized from the rat hippocampus.
Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 microM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mM MnCl2, and 50 micrograms/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2-4 days at 4 degrees C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethylaminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1A binding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.
el Mestikawy
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INSERM U.288, Neurobiologie Cellulaire et Fonctionnelle, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
Taussig
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D
Gozlan
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Emerit
M B
MB
Ponchant
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M
Hamon
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M
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D006624:Hippocampus / Q000032:analysis / Q000378:metabolism*; D006358:Hot Temperature; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011985:Receptors, Serotonin / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013764:Tetrahydronaphthalenes / Q000378:metabolism; D014020:Tissue Extracts / Q000378:metabolism
D011985:Receptors, Serotonin; D013764:Tetrahydronaphthalenes; D014020:Tissue Extracts; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin