Characterization of the rabbit renal Na(+)-dicarboxylate cotransporter using antifusion protein antibodies.
Polyclonal antibodies were prepared against the rabbit renal Na(+)-dicarboxylate cotransporter, NaDC-1. The antibodies were raised in chickens against a fusion protein consisting of a 60-amino acid peptide from NaDC-1 and glutathione S-transferase. These antibodies specifically recognized the fusion protein in Western blots and could immunoprecipitate the full-length NaDC-1 after in vitro translation. The antifusion protein antibodies specifically recognized a protein of 63 kDa in rabbit renal brush-border membrane vesicles (BBMV), similar to the predicted mass of 66 kDa. Two proteins of 57 and 115 kDa were recognized in rabbit intestinal brush-border membranes. A protein of 66 kDa was recognized in Xenopus oocytes injected with NaDC-1 cRNA. Enzymatic deglycosylation of rabbit renal BBMV resulted in a decrease in mass by 11 kDa, consistent with N-glycosylation at a single site. Site-directed mutagenesis of the two consensus sequences for N-glycosylation in the NaDC-1 cDNA showed that Asn-576, located near the COOH-terminal, is glycosylated. The nonglycosylated mutant of NaDC-1 exhibited 50% of wild-type succinate transport activity when expressed in Xenopus oocytes, suggesting that glycosylation is not essential for function. The revised secondary structure model of NaDC-1 contains 11 putative transmembrane domains and an extracellular glycosylated COOH-terminal.
Pajor
A M
AM
Department of Physiology, University of Arizona, College of Medicine, Tucson 85724, USA.
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D000818:Animals; D000906:Antibodies / Q000276:immunology; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D002645:Chickens; D029382:Dicarboxylic Acid Transporters; D005982:Glutathione Transferase / Q000235:genetics* / Q000276:immunology; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D029363:Organic Anion Transporters, Sodium-Dependent; D017510:Protein Folding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D027981:Symporters; D014981:Xenopus
D000906:Antibodies; D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D008565:Membrane Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D011993:Recombinant Fusion Proteins; C092524:SLC13A2 protein, human; D027981:Symporters; D005982:Glutathione Transferase