Purification of the sodium- and chloride-coupled glycine transporter from central nervous system.
We have recently developed a reconstitution assay which allows the rapid determination of sodium- and chloride-dependent glycine transport activity of many fractions (López-Corcuera, B., and Aragón, C. (1989) Eur. J. Biochem. 181, 519-524). In this paper we report the purification of the sodium- and chloride-coupled glycine transporter from pig brain stem. Transporter is solubilized from plasma membrane vesicles with 2% cholate and purified by sequential chromatography on phenyl-Sepharose, wheat germ agglutinin-Sepharose, and hydroxylapatite columns, followed by a 5-20% sucrose density gradient fractionation. Taking into account the inactivation suffered by the transporter, a final increase in specific activity of about 450-fold is achieved. Although two polypeptides with apparent molecular masses of 100 and 37 kDa are progressively enriched during the chromatographic steps, only the 100-kDa band comigrates with transport activity along the density gradient. This band is finally isolated to apparent homogeneity in the active fractions. We conclude that the 100-kDa band represents the glycine transporter. Finally, the pure transporter can be reconstituted into liposomes, retaining the absolute dependence on sodium and chloride gradients, the electrogenicity, the glycine affinity, the substrate specificity, and the sensitivity to group-selective modifiers characteristic of the native transporter.
López-Corcuera
B
B
Departamento de Biología Molecular, Facultad de Ciencias, Universidad Autónoma, Madrid, Spain.
Vázquez
J
J
Aragón
C
C
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001692:Biological Transport; D001933:Brain Stem / Q000737:chemistry*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D050487:Glycine Plasma Membrane Transport Proteins; D008081:Liposomes / Q000378:metabolism; D009994:Osmolar Concentration; D012964:Sodium / Q000378:metabolism*; D013552:Swine; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D002712:Chlorides; D050487:Glycine Plasma Membrane Transport Proteins; D008081:Liposomes; D005680:gamma-Aminobutyric Acid; D012964:Sodium