Molecular properties of 5-hydroxytryptamine3 receptor-type binding sites purified from NG108-15 cells.
5-Hydroxytryptamine3 (5-HT3) receptor-type binding sites were solubilised from NG108-15 mouse neuroblastoma x rat glioma hybrid cells using five different detergents [n-octyl-beta-D-glucoside, Triton X-100, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), sodium cholate, and deoxycholate] and the solubilisation efficiencies compared. The equilibrium binding, kinetic properties, and pharmacological profile of solubilised binding sites were similar to those of 5-HT3 receptor-type binding sites (5-HT3R) in membrane preparations determined using [3H]GR65630. The solubilised binding sites were purified using an affinity column constructed by coupling the high-affinity antagonist GR119566X to an Affi-Gel 15 resin. The affinity of purified 5-HT3R for [3H]-GR65630 was reduced threefold compared to the crude soluble preparation, but the pharmacological profile was similar. The sedimentation coefficient of the purified protein (11S, detergent: CHAPS) was determined by sucrose density gradient centrifugation. The apparent molecular mass of the detergent/binding site complex (370 kDa) was determined by size exclusion chromatography in the presence of n-dodecyl-beta-D-maltoside. Gel electrophoresis of the purified protein revealed bands at apparent molecular masses of 36, 40, 50, and 76 kDa. Electron microscopy of the negatively stained purified protein showed the presence of round particles of 8-9 nm diameter with a 2-nm stained pit in the centre, closely resembling the doughnut shapes described for nicotinic acetylcholine receptors.
Boess
F G
FG
MRC Molecular Neurobiology Unit, MRC Centre, Cambridge, England.
Lummis
S C
SC
Martin
I L
IL
D000818:Animals; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005910:Glioma / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D005960:Glucosides; D007093:Imidazoles / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D051379:Mice; D009447:Neuroblastoma / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D051381:Rats; D011985:Receptors, Serotonin / Q000737:chemistry* / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D014316:Tritium; D014407:Tumor Cells, Cultured
D003902:Detergents; D005960:Glucosides; D007093:Imidazoles; D007211:Indoles; D011985:Receptors, Serotonin; D014316:Tritium; C059068:GR 65630; C018619:octyl-beta-D-glucoside; D012967:Sodium Dodecyl Sulfate