Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P46098 (5-HT3 receptor)
 2,290 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Molecular properties of 5-hydroxytryptamine3 receptor-type binding sites purified from NG108-15 cells. 140 14

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.
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PMID:Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells. 238 Jan 74

1. We demonstrate, for the first time, the purification of the 5-hydroxytryptamine3 (5-HT3) receptor from a native tissue source, pig cerebral cortex. 2. From a range of detergents, the non-ionic detergent Triton X-100 was demonstrated to exhibit the least inhibition of [3H]-(S)-zacopride binding to membrane bound 5-HT3 receptors from pig cerebral cortex at concentrations above its critical micellular concentration (CMC). This detergent was therefore selected to solubilize 5-HT3 binding sites from homogenates of pig cerebral cortex. Maximum yield (43.8 +/- 3.7%, mean +/- s.e.mean, n = 13) was obtained with Triton X-100 at 0.4% (22.1 x CMC). Radioligand binding studies with [3H]-(S)-zacopride indicated that the solubilized 5-HT3 receptor displayed near identical pharmacology to the membrane bound receptor (the correlation coefficient (r) between the pKi values of structurally unrelated compounds competing for [3H]-(S)-zacopride binding in the membrane bound and solubilized 5-HT3 receptor preparations was 0.99, Bmax = 20.7 +/- 4.2 fmol mg(-1) protein, Kd = 1.57 +/- 0.53 nM, mean +/- s.e.mean, n = 6). 3. Solubilized (0.4% Triton X-100) 5-HT3 receptors were affinity purified using Affi-Gel 15 coupled to the high affinity 5-HT3 receptor ligand GR119566X. Radioligand binding studies indicated that the pharmacological profile of the affinity purified 5-HT3 receptor, assessed using ligands with a range of affinities spanning 3 orders of magnitude, was similar to that in both crude homogenates (r = 0.85) and solubilized 5-HT3 receptor sites (r = 0.85) from pig brain. The specific activity for the purified 5-HT3 receptor overlapped the theoretical specific activity of the receptor (Bmax = 3.27 +/- 1.41 and 5.35 +/- 2.33 nmol mg(-1) protein, assessed by saturation and competition studies respectively, mean +/- s.e.mean, n = 3-4), which indicated a 60000-100000 fold purification of the membrane bound receptor. 4. Under non-reducing conditions, samples of the affinity purified protein failed to enter a 10% separating gel in SDS-PAGE analysis, indicating a molecular mass for the receptor complex of > 200 kDa. Further investigation of the non-reduced purified protein with a 7.5% separating gel gave a mass for the complex of approximately 279 kDa. Under reducing conditions, SDS-PAGE analysis of the affinity purified 5-HT3 receptor resulted in 3-6 silver stained bands at apparent molecular masses of 37, 44-50, 52, 57-61, 63 and 65-71 kDa (n = 12). Unlike protein bands at 45, 50, 60 and 66 kDa, the bands corresponding to proteins of 52, 57, 63 and 71 kDa consistently gave no reaction with an antiserum specific for the cloned A subunit of the 5-HT3 receptor in both a modified dot blot procedure and a Western blot procedure (n = 2-5). 5. We conclude that we have purified the 5-HT3 receptor from pig brain to homogeneity and suggest this may contain non-5-HT3-A receptor subunit(s).
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PMID:Purification of 5-hydroxytryptamine3 receptors from porcine brain. 937 61