Pharmacological and biochemical characterization of rat hippocampal 5-hydroxytryptamine1A receptors solubilized by 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS).
Rat hippocampal 5-hydroxytryptamine1A (5-HT1A) binding sites were solubilized with a yield of 34% using 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS, 10 mM) as detergent. Kinetic analyses of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding indicated that the 5-HT1A sites exhibit the same properties in the soluble form as in the membrane-bound form. Furthermore, a positive correlation (r = 0.988) was found between the respective pIC50 values of a series of agonists and antagonists to inhibit [3H]8-OH-DPAT binding to either soluble or membrane-bound 5-HT1A sites. Gel filtration through Sephacryl S-400 as well as chromatography on wheat germ agglutinin (WGA)-agarose did not affect the modulation by guanine nucleotides (5'-guanylylimidodiphosphate) of [3H]8-OH-DPAT binding which suggests that the 5-HT1A binding subunit is a glycoprotein tightly attached to a G protein even in its soluble form. The [3H]8-OH-DPAT binding material eluted from Sephacryl S-400 had an apparent molecular mass of 155 kilodaltons, as expected from a heterodimer with one binding subunit (approximately 60 kilodaltons) and one G protein (approximately 80 kilodaltons). Marked enrichment in 5-HT1A binding sites relative to other soluble proteins was found in the peak fractions eluted from Sephacryl S-400 (by sixfold) and WGA-agarose (by 26-fold) columns, suggesting that these chromatographic steps might be of interest for the purification of central 5-HT1A receptors.
el Mestikawy
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S
INSERM U. 288, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
Cognard
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C
Gozlan
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H
Hamon
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M
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000818:Animals; D002413:Cations, Divalent; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D002793:Cholic Acids; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006624:Hippocampus / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D009711:Nucleotides / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011985:Receptors, Serotonin / Q000187:drug effects / Q000378:metabolism*; D012995:Solubility; D013696:Temperature; D013764:Tetrahydronaphthalenes / Q000378:metabolism
D002413:Cations, Divalent; D002793:Cholic Acids; D003902:Detergents; D009711:Nucleotides; D011985:Receptors, Serotonin; D013764:Tetrahydronaphthalenes; D006165:Guanylyl Imidodiphosphate; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate