Immunodetection and characterization of proteins implicated in renal sodium/phosphate cotransport.
Polyclonal antibodies raised against the 14-amino acid C-terminal portion of the rabbit renal brush-border membrane Na+/Pi cotransporter, as deduced from the nucleotide sequence of the cloned NaPi-1 gene, were used for Western blot analysis of renal brush-border membrane proteins 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 beef were specifically labelled. The affinity of the antibodies was much greater, however, for the proteins of the rat and rabbit than for those of the beef. The rat 65-kDa antigen was readily detected in brush-border membranes isolated from kidney cortex, but was absent from the basolateral membrane and the cytosolic and microsomal fractions of this tissue, in agreement with the subcellular localization of the Na+/Pi cotransporter. This antigen was however several-fold more abundant in the juxtamedullary portion of the cortex than in the outer portion. Despite a strong stimulation in phosphate transport, a low-phosphate diet had little influence on the amount of antigen detected. An additional peptide-displaceable band corresponding to a protein of 250 kDa appeared when beta-mercaptoethanol was omitted during electrophoresis, in agreement with the possibility that disulfide bonds may be involved in the regulation of renal phosphate transport activity.
Delisle
M C
MC
Département de chimie-biochemie, Université du Québec à Montréal, Canada.
Boyer
C
C
Vachon
V
V
Giroux
S
S
Béliveau
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D002417:Cattle; D007672:Kidney Cortex / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000276:immunology; D010710:Phosphates / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism*; D050608:Sodium-Phosphate Cotransporter Proteins; D013347:Subcellular Fractions / Q000378:metabolism; D027981:Symporters
D002352:Carrier Proteins; D008565:Membrane Proteins; D010455:Peptides; D010710:Phosphates; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D012964:Sodium