Characterization of BADS-binding proteins in epithelial plasma membranes

Summary When a fluorescent stilbene was added to epithelial plasma membrane suspension the emission spectrum showed a broad peak containing overlapping emissions resulting from different adducts. By focusing on a specific emission wavelength a common site having a dissociation constant of 5m was calculated in the rat kidney, small intestine, pancreatic islets and shark rectal gland. This binding could be displaced by loop diuretics, (e.g., furosemide with an IC₅₀ of 40 m), DIDS (k _i 1 m) and thiocyanate. These results pose certain questions such as: (i) whether the evidence for multiple peaks are due to specific interactions representing multiple binding affinities and (ii) whether the binding of stilbene and the observed displacement can be identified on a specific protein. Separating the proteins present in the purified basolateral and brush-border membranes by SDS-PAGE, transfer of these proteins onto introcellulose paper and labeling of the nitrocellulose strips by radioactive BADS (4-benzamido-4 aminostilbene-2-2 disulphonic acid) and bumetanide could identify labeled proteins. These experiments showed that whereas some proteins bound either BADS or bumetanide, one protein with a molecular weight of 100 or 130,000 D appeared to bind both. This protein was found on the basolateral membrane in the rat kidney cortex and medulla and the shark rectal gland and in the basolateral and brush-border membranes of the small intestine. Displacement of the protein-bound stilbene by loop diuretics could not be quantitated on the nitrocellulose transfer strips for this protein. Antibodies raised against the cytoplasmic fragment of band 3 reacted with the stilbene-labeled 100–130,000 D proteins indicating sufficient immuno-cross-reactivity between the separate species. These experiments involving binding of BADS and bumetanide and cross-reactivity with the human band 3 antibody suggest that these kilodalton proteins could structurally resemble human band 3.