Reconstitution and characterization of a Na+/Pi co-transporter protein from rabbit kidney brush-border membranes.
A protein with Na+/Pi co-transporter activity has been extracted from rabbit brush-border membranes with chloroform/methanol and purified by hydroxyapatite chromatography. The protein has been incorporated by the dilution method into liposomes formed from different types and ratios of lipids. The greatest reconstitution has been achieved into liposomes prepared from cholesterol (20%), phosphatidylcholine (20%), phosphatidylethanolamine (30%) and phosphatidylserine (30%) (CH/PC/PE/PS). Pi uptake by these proteoliposomes had the following characteristics: (i) the initial rate was markedly greater in the presence of an inwardly directed Na+ gradient (600 pmol/10 s per mg) than with a K+ gradient (65 pmol/10 s per mg); (ii) maximal uptake was increased 8-fold above the equilibrium value ('overshoot') when a Na+ gradient was applied; (iii) Pi was not merely bound to proteoliposomes but was transported intravesicularly; and (iv) Na(+)-dependent Pi uptake was sensitive to the known phosphate transport inhibitors. This first successful attempt of reconstitution of Na+/Pi transport activity into proteoliposomes led us to isolate and characterize physico-chemically the protein responsible. Its isoelectric point was about 5.8, and urea/SDS gel electrophoresis revealed a broad band of molecular mass ranging from 63 to 66 kDa under both reducing and non-reducing conditions. In the native form, the molecular mass analysed by gel filtration was estimated to be 170 +/- 10 kDa, suggesting that the protein is a polymer, probably stabilized by hydrophobic bonds. Endoglycosidase F treatment decreased the molecular mass to approx. 50 kDa. It is postulated that this acidic glycoprotein might represent a subunit of the intact Na+/Pi co-transporter from rabbit kidney brush-border membranes.
Debiec
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INSERM U.64, Hôpital Tenon, Paris, France.
Lorenc
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Ronco
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D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008871:Microvilli / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D011817:Rabbits; D050608:Sodium-Phosphate Cotransporter Proteins; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters
D002352:Carrier Proteins; D008081:Liposomes; D010710:Phosphates; D010743:Phospholipids; D011510:Proteolipids; D050608:Sodium-Phosphate Cotransporter Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; C020485:proteoliposomes