Reconstitution of the renal brush-border membrane sodium/phosphate co-transporter.
A simple and rapid procedure was developed for the reconstitution of Na(+)-dependent phosphate-transport activity from bovine kidney brush-border membranes. The phosphate transporter appears to be particularly sensitive to extraction conditions. To prevent its inactivation, the phosphate carrier was solubilized in a buffer containing its substrates, Na+ and phosphate, CHAPS, dithiothreitol, brush-border membrane lipids and glycerol. The uptake of phosphate by reconstituted vesicles was strongly stimulated by the presence of a transmembrane Na+ gradient. This stimulation was abolished when the Na+ gradient was dissipated by monensin. The affinity of the carrier for phosphate was similar in proteoliposomes and in brush-border membrane vesicles (apparent Kt = 40 microM). The transporter was also stimulated by the presence of a high concentration of phosphate on the trans side of the membrane. The reconstituted transport activity was inhibited by arsenate, a known inhibitor of phosphate transport. However, the bovine phosphate carrier, intact or reconstituted, was much less sensitive to inhibition by phosphonoformic and phosphonoacetic acids than were those of other species studied so far. SDS/PAGE revealed that only a small number of brush-border membrane proteins were incorporated into the proteoliposomes. This reconstitution procedure should be useful for the purification and identification of the carrier protein.
Vachon
V
V
Départment de chimie-biochimie, Université du Québec à Montréal.
Delisle
M C
MC
Laprade
R
R
Béliveau
R
R
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D017245:Foscarnet; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D050608:Sodium-Phosphate Cotransporter Proteins; D013379:Substrate Specificity; D027981:Symporters
D002352:Carrier Proteins; D008565:Membrane Proteins; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D017245:Foscarnet; D008985:Monensin; D010746:Phosphonoacetic Acid