Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain.
The sodium- and potassium-coupled L-glutamate transporter from rat brain has been purified to near homogeneity by reconstitution of transport as an assay, assuming that inactivated and active transporters cochromatograph. The purification steps involve lectin chromatography of the membrane proteins solubilized with 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), fractionation on hydroxylapatite, and ion-exchange chromatography. The specific activity is increased 30-fold. The actual purification is higher since 3-5-fold inactivation occurs during the purification. The efficiency of reconstitution was about 20%. The properties of the pure transporter are fully preserved. They include ion dependence, electrogenicity, affinity, substrate specificity, and stereospecificity. Sodium dodecyl sulfate-polyacrylamide electrophoresis revealed one main band with an apparent molecular mass of around 80 kDa and a few minor bands. Comparison of polypeptide composition with L-glutamate transport activity throughout the fractionation procedure reveals that only the 80-kDa band can be correlated with activity. The GABA transporter, which has the same apparent molecular mass (Radian et al., 1986), is separated from it during the last two purification steps. Immunoblot experiments reveal that the antibodies against the GABA transporter only reacted with fractions exhibiting GABA transport activity and not with those containing the glutamate transporter. We conclude that the 80-kDa band represents the functional sodium- and potassium-coupled L-glutamate transporter.
Danbolt
N C
NC
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Pines
G
G
Kanner
B I
BI
D027322:Amino Acid Transport System X-AG; D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D050485:GABA Plasma Membrane Transport Proteins; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism; D027361:Organic Anion Transporters; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D014909:Wheat Germ Agglutinins
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D006023:Glycoproteins; D007473:Ion Channels; D008081:Liposomes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; D014909:Wheat Germ Agglutinins; D012964:Sodium; D011188:Potassium