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.

journal_name

Neurosci Lett

journal_title

Neuroscience letters

authors

Lambeng N,Grossmann M,Chatelain P,Fuks B

doi

10.1016/j.neulet.2005.12.059

keywords:

subject

Has Abstract

pub_date

2006-05-01 00:00:00

pages

107-12

issue

1-2

eissn

0304-3940

issn

1872-7972

pii

S0304-3940(05)01465-5

journal_volume

398

pub_type

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