Abstract
The novel antiepileptic agent gabapentin (Neurontin) binds with high affinity to the α2δ subunit of a voltage-dependent Ca2+channel. We report here a simple purification scheme for detergent-solubilized α2δ subunits from porcine brain. This involves sequential chromatography on Q-Sepharose, Cu2+-charged iminodiacetic acid–Sepharose, wheat germ lectin–agarose, and Mono Q. The purified protein was essentially homogeneous by SDS–polyacrylamide gel electrophoresis with a subunitMrof 145,000. Using [3H]gabapentin as the radiolabeled tracer and (S)-3-isobutyl γ-aminobutyric acid to define nonspecific binding, the overall purification factor was 2760-fold and the apparent yield 26.6%. We also developed and validated a novel binding assay for α2δ Ca2+channel subunits using the ligand pairl-[3H]leucine/l-isoleucine. Evenin binding assays of crude brain membrane fractions, [3H]leucine proved to be remarkably stable and specific for the α2δ Ca2+channel subunit. [3H]Leucine offers several advantages over custom-labeled [3H]gabapentin: it has a higher specific activity, is relatively inexpensive, and is available from commercial sources.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
