Reconstitution and purification of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain.
The sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain has been highly purified. Synaptic plasma membranes from rat brain were extracted with cholate in the presence of 10% ammonium sulfate. The soluble extract was incorporated into liposomes consisting of asolectin and crude brain lipids. Brain lipids markedly enhanced the transport activity. The resulting proteoliposomes catalyzed sodium- and chloride-coupled gamma-aminobutyric acid transport which, in the presence of internal potassium, was greatly (up to 20-fold) stimulated by valinomycin. Using this transport of the reconstituted system as an assay, the transporter was purified by the following steps. The cholate extract was fractionated by ammonium sulfate. The activity was not precipitated by 50% but could be precipitated by 70% ammonium sulfate. The cholate and ammonium sulfate were removed on a Sephadex G-50 column. Subsequently, the transporter was partially purified on DEAE-cellulose in a mixture of Triton X-100 and octyl glucoside. The active fractions were chromatographed on a hydroxylapatite column in the presence of Triton X-100. Although the increase in specific activity was only up to 100-fold, this was due to partial inactivation. The actual purification was at least 1000-fold. The purified transporter exhibited the same features of the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity, and a similar affinity. The sodium dodecyl sulfate gel pattern indicated that a major protein ran as a 24-kDa band. This band may represent the gamma-aminobutyric acid transporter.
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification*; D002712:Chlorides / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D010455:Peptides / Q000032:analysis; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D012995:Solubility; D014634:Valinomycin / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D003902:Detergents; D010455:Peptides; D014634:Valinomycin; D005680:gamma-Aminobutyric Acid; D012964:Sodium