Hydrodynamic properties and immunological identification of the sodium- and chloride-coupled glycine transporter.
We have recently reported the purification of the native sodium- and chloride-coupled glycine transporter from pig brain stem (López-Corcuera, B. Vázquez, J., and Aragón, C. (1991) J. Biol. Chem. 266, 24809-24814). This preparation is essentially homogeneous and contains a unique 100-kDa polypeptide based on electrophoretic migration under denaturing conditions. In this paper we report the hydrodynamic characterization of the native transporter, solubilized in two different detergents, as well as the immunological identification of the protein. On the basis of results obtained from size-exclusion chromatography, we calculated the Stokes radii of transporter 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and transporter-cholate complexes to be 5.5 and 6.0 nm, respectively. In addition, from H2O/D2O sucrose density gradient sedimentation analysis, we calculated the molecular weight of protein-detergent complexes to be 115,000 and 160,000 in CHAPS and cholate, respectively, and the molecular weight of the protein moiety as 86,000. Finally, polyclonal antibodies raised against the 100-kDa polypeptide were found to immunoprecipitate specifically glycine transport activity. Taken together, the results reported herein corroborate the identity of the 100-kDa band as the sodium- and chloride-coupled glycine transporter and also suggest that in its native state the transporter is a monomeric protein.
López-Corcuera
B
B
Departamento de Biología Molecular, Facultad de Ciencias, Universidad Autónoma, Madrid, Spain.
Alcántara
R
R
Vázquez
J
J
Aragón
C
C
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D000941:Antigens / Q000276:immunology; D001933:Brain Stem / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002712:Chlorides / Q000378:metabolism*; D019826:Cholic Acid; D002793:Cholic Acids / Q000378:metabolism; D002850:Chromatography, Gel; D003903:Deuterium; D050487:Glycine Plasma Membrane Transport Proteins; D007106:Immune Sera / Q000276:immunology; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D012964:Sodium / Q000378:metabolism*; D013552:Swine
D026921:Amino Acid Transport Systems, Neutral; D000941:Antigens; D002352:Carrier Proteins; D002712:Chlorides; D002793:Cholic Acids; D050487:Glycine Plasma Membrane Transport Proteins; D007106:Immune Sera; D012964:Sodium; D003903:Deuterium; D019826:Cholic Acid; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate