Chromatographic Analyses of the Serotonin 5-HT 1A Receptor Solubilized from the Rat Hippocampus (original) (raw)

1989, Journal of Neurochemistry

Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 m M 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 S-HTIA sites led to a buffer composed of 50 mM Tris-HC1, 50 p M dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 m M MnClz, and 50 pg/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HTlA sites, compatible with chromatographic analyses for 2-4 days at 4°C. Adsorption and subsequent elution of [3H]S-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-HTIA 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 S-HT,, sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with M, close to 60,000. Key Words: Hippocampus-[3H]S-Hydroxy-2 -(di -n -propylamino)tetralin -5 -Hydroxytrypta-minelA receptor-Solubilization-Affinity chromatography. El Mestikawy S. et al. Chromatographic analyses of the serotonin 5-HTIA receptor solubilized from the rat hippocampus. Abbreviations used: Brex, bovine brain extract; BSA, bovine serum albumin; CHAPS, 3-[3-(cholamidopropyI)dirnethylammonio]-Ipropane sulfonate; CHS, cholesteryl hemisuccinate; DEAE, diethylaminoethyl; DTT, dithiothreitol; EDAC, I-ethyl-3-(3-dimethyI-arninopropy1)carbodiimide; G protein, guanine nucleotide regulatory protein; 5-HT, 5-hydroxytryptamine or serotonin; kDa, kilodalton; 8-MeO-N-PBAT, 8-methoxy-2-[(N-propyl,N-butylamino)aminoltetralin: NAG, N-acetylglucosamine; NP-40, Nonidet P-40; ODG, octyl-P-D-glucopyranoside; OD, optical density; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; PAGE, polyacrylarnide gel electrophoresis; PEI, polyethylenimine; PIPES, I ,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate: TES, N-tris(hydroxymethyl)rnethyl-2-aminoethanesulfonic acid; WGA, wheat germ agglutinin.