Purification, pharmacological characterization and photoaffinity labeling of sigma receptors from rat and bovine brain
| Autor: | Randall B. Murphy, David I. Schuster, Frank J. Arnold |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Male
Receptor complex Binding Sites Photoaffinity labeling Molecular mass Chemistry General Neuroscience Sigma receptor Rats Sepharose Phenazocine Piperidines Biochemistry Affinity chromatography Cerebellum Dopamine Agonists Animals Haloperidol Receptors sigma Cattle Neurology (clinical) Binding site Receptor Molecular Biology Developmental Biology |
| Zdroj: | Brain Research. 670:14-28 |
| ISSN: | 0006-8993 |
| DOI: | 10.1016/0006-8993(94)01123-y |
| Popis: | The sigma receptor/binding site, found in the brain and periphery, binds haloperidol, (+)-benzomorphans, N-propyl-3-(3-hydroxyphenyl)-piperidine (3-PPP) and certain atypical neuroleptics with high affinity. We have succeeded in ca. 6,000-fold purification of protein(s) from rat and bovine cerebellum which display pharmacology characteristic of the sigma receptor. This purification was achieved by affinity chromatography using a Sepharose gel linked to a new high-affinity ligand, (S)-3-(3-methoxyphenyl)-3'-oxo-3'-phenyl-N-propylpiperidine, an analog of (S)-3-PPP. Elution of the affinity column with haloperidol afforded material which, after reconstitution into bimolecular lipid vesicles, was pharmacologically characterized by specific radioligand binding assays using [3H]haloperidol combined with competitive displacement using appropriate selective ligands. Comparison of the relative rank orders of potency of the ligands in these selective sigma receptor assays corresponded well with values obtained with tissue homogenates. The observed enantioselectivity for the binding of SKF-10,047 and cyclazocine suggests that the material purified corresponds to the sigma 1 receptor subtype. SDS-PAGE indicated that the purified material consisted of two bands of approximate molecular masses 65 and 63 kilodaltons. Photoaffinity labeling of the affinity-purified receptor with [3H]azido-DTG led to incorporation of the label into material of molecular mass 50-70 kDa, by slicing of SDS gels, while similar photolabeling of crude cerebellar homogenates led to exclusive labeling of a 29 kDa polypeptide, as found previously using other tissues. Molecular sizing under non-denaturing conditions indicated the photolabeled species is a labile large receptor complex of mass ca. 300-500 kDa which gradually breaks down upon standing at -80 degrees C into the lower mass (50-70 kDa) material. The sigma receptor ligand binding subunit, which appears to be of the sigma 1 subtype, appears to be contained within the 29 kDa polypeptide, which may be a subunit of the 63-65 kDa protein, which in turn appears to be a component of a much larger receptor complex. It further appears that the 29 kDa polypeptide is readily dissociable from a larger photolabeled sigma receptor complex in tissue homogenates, but does not dissociate from the photolabeled affinity-purified CHAPS-solubilized sigma receptor. |
| Databáze: | OpenAIRE |
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