MOLECULAR-SIZE OF THE FUNCTIONAL COMPLEX AND PROTEIN SUBUNITS OF THE RENAL PHOSPHATE SYMPORTER

The oligomeric structure of the rabbit renal brush-border membrane sod ium/phosphate cotransporter was examined with the radiation inactivati on and fragmentation technique. The size of its functional complex (it s ''radiation inactivation size'') was estimated from the rate of deca y of its sodium-dependent transport activity as a function of the radi ation dose. A radiation inactivation size of 223 +/- 42 kDa was obtain ed. The polypeptide constituting the monomeric unit of the Na1+/P-i sy mporter was detected by immunoblotting with polyclonal anti-peptide an tibodies directed against the 14 amino acid C-terminal portion of the symporter molecule. Its apparent molecular size estimated by compariso n with standards following SDS-polyacrylamide gel electrophoresis was 64 000. This value is in good agreement with its known molecular mass of 51 797 Da calculated from the amino acid sequence deduced from the nucleotide sequence of its gene since this protein is probably glycosy lated. The loss of labeling intensity of the polypeptide of M(r) = 64 000 was also measured as a function of radiation dose. The molecular s ize calculated from these data (its ''target size'') was 165 +/- 20 kD a. The target size estimated for the rat phosphate cotransporter was 1 84 +/- 46 kDa, and its previously reported radiation inactivation size was 234 +/- 14 kDa. These results strongly suggest that the renal Na1 +/P-i cotransporter exists as an oligomeric protein; probably a homote tramer. The fact that the values obtained for the target size are abou t 3/4 those obtained for the radiation inactivation size of these cotr ansport proteins indicates that their subunits are closely associated since most of their subunits appear to be fragmented by a single ioniz ing radiation hit.