IMMUNODETECTION AND CHARACTERIZATION OF PROTEINS IMPLICATED IN RENAL SODIUM-PHOSPHATE COTRANSPORT

Polyclonal antibodies raised against the 14-amino acid C-terminal port ion of the rabbit renal brush-border membrane Na+/P(i) cotransporter, as deduced from the nucleotide sequence of the cloned NaPi-1 gene, wer e used for Western blot analysis of renal brush-border membrane protei ns from rat, rabbit and beef. Proteins of 65 kDa from the rat, 64 kDa from the rabbit, and 38, 66, 77, 92, 110, 176 and 222 kDa from the bee f were specifically labelled. The affinity of the antibodies was much greater, however, for the proteins of the rat and rabbit than for thos e of the beef. The rat 65-kDa antigen was readily detected in brush-bo rder membranes isolated from kidney cortex, but was absent from the ba solateral membrane and the cytosolic and microsomal fractions of this tissue, in agreement with the subcellular localization of the Na+/P(i) cotransporter. This antigen was however several-fold more abundant in the juxtamedullary portion of the cortex than in the outer portion. D espite a strong stimulation in phosphate transport, a low-phosphate di et had little influence on the amount of antigen detected. An addition al peptide-displaceable band corresponding to a protein of 250 kDa app eared when beta-mercaptoethanol was omitted during electrophoresis, in agreement with the possibility that disulfide bonds may be involved i n the regulation of renal phosphate transport activity.