Solubilization and immunopurification of rat brain synaptic vesicle protein 2A with maintained binding properties.
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.
Lambeng
N
N
UCB S.A., CNS In Vitro Pharmacology, Building R4, Chemin du Foriest, B-1420 Braine-l'Alleud, Belgium. nathalie.lambeng@ucb-group.com
Grossmann
M
M
Chatelain
P
P
Fuks
B
B
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
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