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dc.contributor.advisorAna M. Pajoren_US
dc.creatorHongyan Lien_US
dc.date.accessioned2011-12-20T16:04:36Z
dc.date.available2003-05-21en_US
dc.date.available2011-12-20T16:04:36Z
dc.date.created2003-04-10en_US
dc.date.issued2003-01-28en_US
dc.identifier.otheretd-04102003-012738en_US
dc.identifier.urihttp://hdl.handle.net/2152.3/95
dc.description.abstractIn this dissertation, two cation-coupled transporters were characterized at the molecular level. The CitM transporter from Bacillus subtilis was functionally expressed and characterized in E.coli cells. The human NaSi-1 transporter (hNaSi-1) and mutants were functionally expressed in Xenopus oocytes. Antibodies against hNaSi-1 were used to investigate tissue distribution and N-glycosylation. The roles of two conserved serine residues in the transport function of hNaSi-1 were investigated using site-directed mutagenesis and radiotracer assay. \r\n\r\n CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the Km of CitM for the complex of Mg2+-citrate was about 300 mM in the presence of saturating Mg2+ concentrations. CitM has a high substrate specificity for citrate. Other tested di- and tricarboxylic acids did not significantly inhibit citrate uptakes in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The transport was inhibited in more alkaline but not in acidic transport buffer and also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin, suggesting a proton-coupled transport. Valinomycin did not affect the uptake by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. \r\n\r\nThe low affinity Na+/sulfate cotransporter, hNaSi-1, belongs to a specific gene family of Na+-coupled transporters that includes the high affinity hSUT-1 and the Na+-coupled dicarboxylate (NaDC) transporters. Antibodies directed against a peptide of hNaSi-1 recognized the native protein in renal membranes as well as the recombinant protein expressed in Xenopus oocytes. There is a single N-glycosylation site, Asn-591, located at the extracellular C-terminus in hNaSi-1. Site-directed mutagenesis studies of Ser-260, Ser-288 and the surrounding amino acid residues of hNaSi-1 suggested that these residues are functionally required for hNaSi-1. MTSET inhibition on sulfate uptakes by the four mutants surrounding Ser-260, T257C, T259C, T261C and L263C, was dependent on the cation and substrate used. Since the presence of sodium and sulfate triggers conformational changes during the transport cycle of hNaSi-1, the cation and substrate dependence of MTSET inhibition suggest that these four substituted cysteines move during the transport cycle. Since the four mutated residues are located in TMD-5, this transmembrane domain is also likely to participate in the conformational movement during the transport cycle of hNaSi-1. \r\nen_US
dc.format.mediumelectronicen_US
dc.language.isoengen_US
dc.rightsCopyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works.en_US
dc.subjectWestern blotsen_US
dc.subjectradiotracer uptake assayen_US
dc.subjectPCRen_US
dc.subjectmutagenesisen_US
dc.subjectimmunofluorescenceen_US
dc.subjectbrush border membrane vesiclesen_US
dc.subjectbiotinylationen_US
dc.subjecten_US
dc.titleMolecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1en_US
dc.type.materialtexten_US
dc.type.genredissertationen_US
thesis.degree.namePhDen_US
thesis.degree.levelDoctoralen_US
thesis.degree.grantorThe University of Texas Medical Branchen_US
thesis.degree.departmentPhysiology and Biophysicsen_US
dc.contributor.committeeMemberSteven C. Kingen_US
dc.contributor.committeeMemberSteven A. Weinmanen_US
dc.contributor.committeeMemberLuis Reussen_US
dc.contributor.committeeMemberJoel P. Gallagheren_US


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