Factors affecting the stability of the renal sodium/phosphate symporter during its solubilization and reconstitution.
Phosphate is reabsorbed across the brush-border membrane of the proximal tubule by a specific sodium-dependent symporter. Like the other brush-border membrane transport proteins of the kidney, the phosphate carrier remains to be isolated in a functional state. To establish a set of parameters that allow to preserve its biological activity, the phosphate carrier was solubilized under systematically varied conditions and reconstituted into proteoliposomes. Successful reconstitution was achieved only when the extraction buffer contained lipids extracted from the renal brush-border membrane. Glycerol, an osmolyte which reduces the water activity of the solution, was also required. It could however be replaced by 150 mM sodium or potassium phosphate. Below this concentration and in the presence of glycerol, the ionic strength of the solution had little effect on the stability of the transporter, but sodium phosphate could not be replaced by sodium chloride. Phosphate transport in reconstituted vesicles depended on the concentration of detergent and pH of the extraction buffer. Finally, transport activity was increased when solubilization was carried out in the presence of a reducing agent, dithiothreitol. These results should be helpful during the purification and further characterization of the renal phosphate symporter.
Vachon
V
V
Départment de chimie-biochimie, Université du Québec à Montréal, Canada.
Delisle
M C
MC
Giroux
S
S
Laprade
R
R
Béliveau
R
R
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002784:Cholesterol; D002793:Cholic Acids; D004229:Dithiothreitol; D005990:Glycerol; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry*; D008563:Membrane Lipids; D008871:Microvilli / Q000737:chemistry; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D013696:Temperature
D002352:Carrier Proteins; D002793:Cholic Acids; D008563:Membrane Lipids; D010710:Phosphates; D011510:Proteolipids; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; C020485:proteoliposomes; D002784:Cholesterol; D005990:Glycerol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D004229:Dithiothreitol