The amine transporter from bovine chromaffin granules. Partial purification.
We have partially purified the amine transporter from bovine adrenal chromaffin granules in a single step utilizing affinity chromatography. A 5-hydroxytryptamine moiety has been coupled to a Sepharose 4B matrix in a position ortho to the hydroxyl group. When membranes solubilized with sodium cholate are chromatographed on the above matrix a 45,000 Mr polypeptide is highly enriched. The enrichment is dependent on the presence of the proper ligand on the matrix and is inhibited if the column is previously equilibrated with a soluble ligand. Enrichment of the above polypeptide is accompanied by an increase in the specific activity of the transporter as measured by its labeling by 4-azido-3-nitrophenylazo(5-hydroxytryptamine). The ability of reserpine, a competitive inhibitor of binding and transport, to inhibit labeling of the purified transporter correlates well with its known kinetic constants in the native membranes. The polypeptide purified is identical to the one previously identified as the putative transporter based on specific labeling by a photoaffinity label (Gabizon, R., Yetinzon, T., and Schuldiner, S. (1982) J. Biol. Chem. 257, 15145-15150). The results clearly support the contention that the 45,000 Mr peptide is the amine transporter or one of its subunits.
D000588:Amines / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002837:Chromaffin Granules / Q000032:analysis*; D002838:Chromaffin System / Q000032:analysis*; D002846:Chromatography, Affinity / Q000379:methods; D006023:Glycoproteins / Q000276:immunology*; D007700:Kinetics; D008970:Molecular Weight; D012701:Serotonin / Q000031:analogs & derivatives / Q000378:metabolism
D000588:Amines; D002352:Carrier Proteins; D006023:Glycoproteins; D012701:Serotonin; C036823:4-azido-3-nitrophenyl-azo-(5-hydroxytryptamine)