title: Solubilization and immunopurification of rat brain synaptic vesicle protein 2A with maintained binding properties.
abstract: This study reports the solubilization of the rat synaptic vesicle protein SV2A, the brain binding site for the antiepileptic drug levetiracetam (LEV), and its characterization. N-dodecyl-beta-D-maltoside (DDM) was the best detergent at achieving a high percentage of SV2A solubilization and at maintaining the binding characteristics of a tritiated form of a more potent analogue of LEV, [3H]ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide). Scatchard analysis revealed that approximately 25% of SV2A proteins from brain membranes are solubilized by DDM under optimal conditions. Competition binding experiments with a variety of LEV analogues indicated that [3H]ucb 30889 labels the same binding site in both crude homogenates and soluble extracts, with still high stereoselectivity. After immunoprecipitation of SV2A from solubilized rat brain membranes, binding properties of [3H]ucb 30889 to SV2A and association with synaptotagmin I were maintained. The two other isoforms SV2B and SV2C were found to be co-immunoprecipitated with SV2A. The solubilization and immunopurification of SV2A with unmodified ligand affinities and synaptotagmin I interaction provides the starting point for future protein-protein interactions and structural studies.
authors.affiliation: UCB S.A., CNS In Vitro Pharmacology, Building R4, Chemin du Foriest, B-1420 Braine-l'Alleud, Belgium. nathalie.lambeng@ucb-group.com
authors.forename: N
authors.initials: N
authors.lastname: Lambeng
authors.forename: M
authors.initials: M
authors.lastname: Grossmann
authors.forename: P
authors.initials: P
authors.lastname: Chatelain
authors.forename: B
authors.initials: B
authors.lastname: Fuks
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry; D001667:Binding, Competitive; D002540:Cerebral Cortex / Q000737:chemistry* / Q000378:metabolism; D005960:Glucosides / Q000737:chemistry; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008566:Membranes; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011759:Pyrrolidines / Q000737:chemistry; D011869:Radioligand Assay; D051381:Rats; D013501:Surface-Active Agents / Q000737:chemistry*; D014316:Tritium
chemical_list: C487592:(2S)-2-(4-(3-azidophenyl)-2-oxopyrrolidin-1-yl)butanamide; D001386:Azides; D005960:Glucosides; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011759:Pyrrolidines; D013501:Surface-Active Agents; C489618:Sv2a protein, rat; D014316:Tritium; C040358:dodecyl maltoside