IDENTIFICATION AND EPITOPE ANALYSIS OF THE RENAL NA+ P(I) COTRANSPORTPROTEIN USING MONOCLONAL-ANTIBODIES/

Seven monoclonal antibodies (mAbs) were raised against a rabbit renal brush-border glycoprotein (molecular mass, 63-66 kDa), presumably invo lved in Na+/P(i) cotransport, which we had previously purified and rec onstituted in active form in proteoliposomes (Debiec, H., Lorenc, R., and Ronco, P. M. (1992) Biochem. J. 286, 97-102). Antibody specificity for the 63-66-kDa protein was analyzed by enzyme-linked immunosorbent assay and confirmed by Western blotting and immunoaffinity chromatogr aphy of solubilized brush-border membranes (BBM), which both yielded a single 63-66-kDa band. Enzyme-linked immunosorbent assay and immunobl otting of renal cortical cell subfractions localized the immunoreactiv e protein to the brush-border membrane. This location was confirmed by indirect immunofluorescence of kidney cortex sections. Binding of two of the seven mAbs (63A20 and 206A126) to native BBM only occurred whe n the related epitope was exposed in the presence or absence of Na+, r espectively; the other mAbs did not react with native BBM probably bec ause of intramembranous orientation of the epitopes. mAb 63A20 inhibit ed dose-dependently Na+/P(i) cotransport when preincubation of BBM was carried out in the presence of Na+ but did not affect Na+/D-glucose c otransport. Proteoliposomes formed from BBM proteins depleted of the 6 3-66-kDa protein by affinity chromatography with mAb 63A20 showed an 8 5% reduction in Na+/P(i) cotransport, whereas Na+/D-glucose cotranspor t was not modified. These results thus establish that the 63-66-kDa BB M protein is the essential component of the Na+/P(i) cotransport syste m. The present study also provides the first immunologic tools availab le for immunohistochemical localization of the Na+/P(i) cotransporter. Finally, the identification of a functional epitope by mAb 63A20 open s up new ways to explore the molecular aspects of P(i) uptake.