Physicochemical properties of serotonin 5-HT3 binding sites solubilized from membranes of NG 108-15 neuroblastoma-glioma cells.
Specific binding sites with pharmacological properties typical of serotonin 5-HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108-15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield, 50%) could be achieved using the detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris-HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000-g CHAPS extract was saturable and showed characteristics (Bmax = 425 +/- 81 fmol/mg of protein; KD = 0.19 +/- 0.02 nM) closely related to those of membrane-bound sites (Bmax = 932 +/- 183 fmol/mg of protein; KD = 0.60 +/- 0.03 nM). Determination of association (k+1 = 0.17 nM min-1) and dissociation (k-1 = 0.02 min-1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]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-HT3 antagonists (zacopride, ICS 205-930, and MDL 72222) and decrease in the affinity for 5-HT3 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 [3H]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 [3H]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-HT3 receptors. Two soluble materials with apparent molecular masses of approximately 600 and approximately 36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S-400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108-15 cells gave a value of approximately 35 kDa.
INSERUM U. 288, Neurobiologie Cellulaire et Fonctionnelle, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
M C
MC
Miquel
M B
MB
Emerit
F J
FJ
Bolaños
L E
LE
Schechter
H
H
Gozlan
M
M
Hamon
D000818:Animals; D001549:Benzamides / Q000378:metabolism; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002462:Cell Membrane / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents; D005910:Glioma / Q000378:metabolism / Q000473:pathology; D008970:Molecular Weight; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D011985:Receptors, Serotonin / Q000737:chemistry*; D012702:Serotonin Antagonists / Q000378:metabolism; D012995:Solubility; D014407:Tumor Cells, Cultured
D001549:Benzamides; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002793:Cholic Acids; D003902:Detergents; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; C055971:zacopride; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate