Solubilization, purification and pharmacological characterization of bovine striatal dopamine D-2 receptor
| Autor: | Ramwani, Jairam J. |
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| Rok vydání: | 2010 |
| Předmět: | |
| Druh dokumentu: | Diplomová práce |
| Popis: | With the advent of radioligand binding assays, central nervous system dopamine receptors have been well characterized in their membrane bound state. These receptors have been grouped into D-1 and D-2 subclasses on the basis of their relationship to the enzyme adenylate cyclase and affinities for dopamine agonists and antagonists. The dopamine D-2 receptor is considered relevant to the behavioral and pharmacological effects of neuroleptic drugs. The studies presented in this dissertation describe a successful method of solubilization of bovine striatal membrane bound dopamine D-2 receptor. The solubilized receptor exhibited typical pharmacological characteristics to that of membrane bound dopamine D-2 receptor. The rank order potency of agonists and antagonists to displace [³H]spiroperidol binding was the same as those observed with the membrane bound receptor. Analysis of the [³H]spiroperidol/agonist competition curves and the [³H]NPA binding revealed the retention of high and low affinity states of dopamine D-2 receptor in the solubilized preparation. This study demonstrated for the first time, a successful affinity chromatography method for the purification of dopamine D-2 receptor. One cycle affinity purification resulted in a 2000-fold enrichment of dopamine D-2 receptor activity with a recovery of 12% from the membrane-bound state and a specific activity of 169,600 fmol/mg protein (assayed with [³H]spiroperidol). The order of potency of D-2 agonists (N-propylnorapomorphine > N0434 > apomorphine > dopamine) and antagonists (spiroperidol > (+)-butaclamol > domperidone) with a purified preparation was found to be similar to that of the membrane bond or solubilized dopamine D-2 receptor. The adsorption of receptor to the affinity matrix was biospecific as pre-incubation of the solubilized preparation with D-2 receptor agonists or antagonists blocked retention of receptor activity. Elution of receptor was also biospecific as dopaminergic drugs were effective in eluting the bound receptor. Affinity purified preparations should be useful in producing monoclonal antibody to dopamine D-2 receptor and also prove to be important in understanding the molecular events from receptor drug binding to final response. Doctor of Philosophy (PhD) |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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