Purification of 5-hydroxytryptamine3 receptors from porcine brain.
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).
Fletcher
S
S
Department of Pharmacology, The Medical School, University of Birmingham, Edgbaston.
Barnes
N M
NM
D000818:Animals; D001549:Benzamides / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D011485:Protein Binding; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000302:isolation & purification* / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3; D012702:Serotonin Antagonists / Q000378:metabolism; D013552:Swine
D001549:Benzamides; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D012702:Serotonin Antagonists; C055971:zacopride