Interaction of alpha- and beta-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta-subunit |
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Authors: | Okamoto C T Chow D C Forte A J |
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Institution: | Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90089-9121, USA. cokamoto@hsc.usc.edu |
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Abstract: | The assembly of the -subunit of thegastric H-K-ATPase (HK ) with the -subunit of the H-K-ATPase orthe Na-K-ATPase (NaK ) was characterized with two anti-HK monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, inH-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cellstransfected with HK , MAb 2/2E6 was observed to bind to HK onlywhen interactions between - and -subunits were disrupted byvarious denaturants. The epitope for MAb 2/2E6 was mapped to thetetrapeptide S226LHY229 of the extracellulardomain of HK . The epitope for MAb 2G11 was mapped to the eightNH2-terminal amino acids of the cytoplasmic domain ofHK . In transfected MDCK cells, MAb 2G11 could immunoprecipitate HK with -subunits of the endogenous cell surface NaK , as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HK . In HK -transfected LLC-PK1 cells,significant immunofluorescent labeling of HK at the cell surfacecould be detected without postfixation denaturation or in live cells,although a fraction of transfected HK could also becoimmunoprecipitated with NaK . Thus assembly of HK with NaK does not appear to be a stringent requirement for cell surface deliveryof HK in LLC-PK1 cells but may be required in MDCKcells. In addition, endogenous posttranslational regulatory mechanismsto prevent hybrid - heterodimer assembly appear to be compromisedin transfected cultured renal epithelial cells. Finally, theextracellular epitope for assembly-sensitive MAb 2/2E6 may represent aregion of HK that is associated with - interaction. |
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