Partial purification of the sodium- and potassium-coupled L-glutamate transport glycoprotein from rat brain.

The sodium- and potassium-coupled L-glutamate transporter from rat brain has been solubilized with cholate and 10-20-fold purified using Wheat Germ Agglutinin-Sepharose 4B. Transport activity--as determined upon reconstitution of the fraction into liposomes--was retained on the column and eluted by N-acetylglucosamine. When the glycoprotein fraction was depleted of the N-acetylglucosamine and applied to a second round of lectin-chromatography, the L-glutamate transport activity was retained and again could be eluted by the sugar. The transporter activity reconstituted from the glycoprotein fraction exhibited the same features as that in synaptic plasma membranes, including electrogenicity, an absolute dependence on external sodium and internal potassium, affinity and stereospecificity. Furthermore, efflux and exchange properties of the reconstituted preparation were also unchanged by the solubilisation and lectin-chromatography. These observations indicate that the sodium- and potassium-coupled L-glutamate transporter is a glycoprotein and is predominantly reconstituted in the 'right-side-out' conformation.