Purification and identification of the functional sodium- and chloride-coupled gamma-aminobutyric acid transport glycoprotein from rat brain.
Using the reconstitution conditions developed recently (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865) we have now purified the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain to apparent homogeneity. A partially purified transporter preparation was passed over wheat germ agglutinin-Sepharose 6MB and non-bound proteins were washed away. The transport activity, as expressed upon reconstitution of the protein into liposomes, was eluted by a solution containing Triton X-100 and N-acetylglucosamine. The specific transport activity was increased almost 400-fold over that of the crude extract. Taking into account an approximately 2.5-fold inactivation during the lectin column chromatography, the actual purification is about 1000-fold. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the active fractions revealed one band of 80 kDa and small amounts of a band which ran at an apparent molecular mass of 160 kDa. The ratio between the two could be experimentally changed such as, for instance, by lyophilization. Polyclonal antibodies were prepared against the 80-kDa band which also cross-reacted with the 160-kDa band, indicating that the latter apparently represents a dimer form of the first. Using Protein A-Sepharose Cl-4B and the antibody against the 80-kDa band, we were able to quantitatively immunoprecipitate the potential gamma-aminobutyric acid transport activity from a crude transporter preparation. The pure transporter preparation exhibited the same features of the transporter in synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogeneity, affinity, and efflux and exchange properties. We conclude that the 80-kDa band represents the gamma-aminobutyric acid transporter.
D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins; D002712:Chlorides / Q000494:pharmacology; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D050485:GABA Plasma Membrane Transport Proteins; D007163:Immunosorbent Techniques; D008081:Liposomes / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism; D027361:Organic Anion Transporters; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; D005680:gamma-Aminobutyric Acid; D012964:Sodium