Molecular size of the functional complex and protein subunits of the renal phosphate symporter.
The oligomeric structure of the rabbit renal brush-border membrane sodium/phosphate cotransporter was examined with the radiation inactivation and fragmentation technique. The size of its functional complex (its "radiation inactivation size") was estimated from the rate of decay of its sodium-dependent transport activity as a function of the radiation dose. A radiation inactivation size of 223 +/- 42 kDa was obtained. The polypeptide constituting the monomeric unit of the Na1+/Pi symporter was detected by immunoblotting with polyclonal anti-peptide antibodies directed against the 14 amino acid C-terminal portion of the symporter molecule. Its apparent molecular size estimated by comparison 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 deducted from the nucleotide sequence of its gene since this protein is probably glycosylated. The loss of labeling intensity of the polypeptide of M(r) = 64,000 was also measured as a function of radiation dose. The molecular size calculated from these data (its "target size") was 165 +/- 20 kDa. The target size estimated for the rat phosphate cotransporter was 184 +/- 46 kDa, and its previously reported radiation inactivation size was 234 +/- 14 kDa. These results strongly suggest that the renal Na1+/Pi cotransporter exists as an oligomeric protein, probably a homotetramer. The fact that the values obtained for the target size are about 3/4 those obtained for the radiation inactivation size of these cotransport proteins indicates that their subunits are closely associated since most of their subunits appear to be fragmented by a single ionizing radiation hit.
Delisle
M C
MC
Département de chimie-biochimie, Université du Québec à Montréal, Canada.
Giroux
S
S
Vachon
V
V
Boyer
C
C
Potier
M
M
Béliveau
R
R
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D008871:Microvilli / Q000378:metabolism* / Q000528:radiation effects; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D002352:Carrier Proteins; D046911:Macromolecular Substances; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters