IMMUNOELECTRON MICROSCOPY OF EPITOPES ON NA,K-ATPASE CATALYTIC SUBUNIT - IMPLICATIONS FOR THE TRANSMEMBRANE ORGANIZATION OF THE C-TERMINAL DOMAIN

The transmembrane folding of the alpha subunit of Na,K-ATPase was stud ied by using immunoelectron microscopy to determine whether monoclonal antibodies with defined epitopes bind to the extracellular or cytopla smic surface. In double labeling experiments, an antibody and a refere nce marker were bound to purified membrane-associated Na,K-ATPase and were visualized by employing colloidal gold particles of two different sizes. Wheat germ agglutinin and a previously characterized monoclona l antibody were used as control markers for the exoplasmic and cytopla smic surfaces, respectively. Three antibodies, VG4, VG2, and IIC9, una mbiguously bound to the extracellular surface. Previously IIC9 had bee n assigned to the cytoplasmic surface because, in immunofluorescence s tudies, it stained intact cells only when they were detergent-permeabi lized. To investigate the basis for this contradiction, a third assay for sidedness was used: competition binding in solution to right-side- out renal medullary vesicles. IIC9 was found to bind to the extracellu lar surface of sealed vesicles, but only in certain experimental condi tions. It was concluded that IIC9 has an epitope that is not always ac cessible and that in this instance, studies of binding to intact and d etergent-treated cells had given misleading results. An extracellular disposition for all three antibodies is not compatible with existing f olding models, and new models are presented.