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Physicochemical Properties of Serotonin 5‐HT 3 Binding Sites Solubilized from Membranes of NG 108‐15 Neuroblastoma‐Glioma Cells
Author(s) -
Miquel M.C.,
Emerit M. B.,
Bolaños F. J.,
Schechter L. E.,
Gozlan H.,
Hamon M.
Publication year - 1990
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1990.tb04935.x
Subject(s) - chaps , chemistry , membrane , dissociation constant , digitonin , binding site , receptor , chromatography , stereochemistry , biochemistry
Specific binding sites with pharmacological properties typical of serotonin 5‐HT 3 receptors were identified in membranes of the murine hybridoma cell line NG 108‐15, using [ 3 H]zacopride as a ligand. Optimal solubilization of these sites (yield. 50%) could be achieved using the detergent 3–[3–(cholamidopropyl)dimethylammonio]‐1‐propane sul‐fonate (CHAPS) at 24 m M plus 0.5 M NaCl in 25 m M Tris‐HCl, pH 7.4. Specific [ 3 H]zacopride binding to soluble sites in the 100,000‐ g CHAPS extract was saturable and showed characteristics ( B max = 425 ± 81 fmol/mg of protein; K D = 0.19 ± 0.02 n M ) closely related to those of membrane‐bound sites ( B max = 932 ± 183 fmol/mg of protein; K D = 0.60 ± 0.03 n M ). Determination of association ( K +1 =0.17 n M min −1 ) and dissociation ( k ‐1 = 0.02 min −1 ) rate constants for the soluble sites gave a K D value of 0.12 n M , a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC 50 ) of 10 different drugs, the pharmacological profile of [ 3 H]zacopride specific binding sites was essentially the same ( r = 0.99) in the CHAPS‐soluble extract and in cell membranes, although some increase in the affinity for 5‐HT 3 antagonists (zacopride, ICS 205–930, and MDL 72222) and decrease in the affinity for 5‐HT 3 agonists (2‐methyl‐5‐hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS‐soluble extract gave a Svedberg coefficient of 12S for the material with [ 3 H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S‐400 and wheat germ agglutinin‐agarose columns indicated marked enrichment (by 2.5– and 10‐fold, respectively) in [ 3 H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5‐HT 3 receptors. Two soluble materials with apparent molecular masses of ∼600 and ∼36 kDa were found to bind [ 3 H]zacopride specifically in the Sephacryl S‐400 eluate. Interestingly, molecular mass determination by radiation inactivation of [ 3 H]zacopride binding sites in frozen NG 108–15 cells gave a value of ∼35 kDa.