INVOLVEMENT OF DISULFIDE BONDS IN THE RENAL SODIUM PHOSPHATE COTRANSPORTER NAPI-2/

The rat renal brush border membrane sodium/phosphate cotransporter NaP i-2 was analysed in Western blots with polyclonal antibodies raised ag ainst its N-terminal and C-terminal segments. Under reducing condition s, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N- terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) we re detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produc e a given effect in intact membrane vesicles than in solubilized prote ins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decrea sed with increasing concentrations of reducing agents [beta-mercaptoet hanol, dithiothreitol and tris-(2-carboxy-ethyl)phosphine] and was lin early correlated with the amount of p180 detected. The target sizes es timated from the radiation-induced loss of intensity of p40, p70 and p 180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an olig omeric protein in which the subunits are sufficiently close to one ano ther to allow substantial energy transfer between the monomers. When p rotein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, ta rget sizes estimated from the radiation-induced loss of intensity of p 40 and p70 were 74 and 92 kDa respectively, showing the presence of di sulphide bridges in the molecular structure of NaPi-2.