Reconstitution of the renal brush-border membrane sodium/phosphate co-transporter

Abstract

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.

Speed‐mapping of arsenic distribution in the tissues of earthworms inhabiting arsenious soil.

“…Arsenate, the pentavalent oxyanion, is structurally similar to phosphate (Wetterhahn-Jennette, 1981) and its toxicity is largely due to interactions with phosphate transport and metabolism. For example: arsenate competeswith phosphate in the sodium-dependent cotransport in bovine renal brush-border membranes (Vachon et al, 1991); arsenate inhibits ATP synthesis by uncoupling oxidative phosphorylation (Clarkson, 1993). Furthermore, Meharg and McNair (1992) showed that the phenotypic expression of arsenate-tolerance in the grass, Holcus lanatus, is suppressed phosphate uptake; Davenport and Peryea (1991) showed that the application of phosphate fertilizers to lead arsenate-contaminated soils increases arsenic mobility and phytotoxicity.…”

Molecular and ionic mimicry and the transport of toxic metals

“…Experiments in osteosarcoma cells (ROS 17/2.8; Caverzasio et al, 1988), bovine and rabbit renal brush border membrane vesicles (Schali et al, 1986;Vachon et al, 1991), vesicles from the matrix of chicken cartilage (Montessuit et al, 1991), and the luminal plasma membrane of rat intestine (Ishizawa et al, 1990) show that arsenate will compete with phosphate at the site of the sodium-dependent phosphate co-transporter. Additionally, arsenate has been shown to inhibit competitively phosphate transport at the site of an uncharacterized sodium-independent transporter (Azzarolo et al, 1991;Quamme et al, 1989).…”