Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Sep 1;278(Pt 2):543–548. doi: 10.1042/bj2780543

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

V Vachon 1, M C Delisle 1, R Laprade 1, R Béliveau 1
PMCID: PMC1151379  PMID: 1832858

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.

Full text

PDF
543

Images in this article

547Fig. 5.
on p.547

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AMES B. N., DUBIN D. T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem. 1960 Mar;235:769–775. [PubMed] [Google Scholar]
  2. Ambudkar S. V., Maloney P. C. Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis. J Biol Chem. 1986 Aug 5;261(22):10079–10086. [PubMed] [Google Scholar]
  3. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. Bindels R. J., van den Broek L. A., van Os C. H. Effect of pH on the kinetics of Na+-dependent phosphate transport in rat renal brush-border membranes. Biochim Biophys Acta. 1987 Feb 12;897(1):83–92. doi: 10.1016/0005-2736(87)90317-8. [DOI] [PubMed] [Google Scholar]
  5. Booth A. G., Kenny A. J. A rapid method for the preparation of microvilli from rabbit kidney. Biochem J. 1974 Sep;142(3):575–581. doi: 10.1042/bj1420575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Béliveau R., Strevey J. The sodium gradient induces conformational changes in the renal phosphate carrier. J Biol Chem. 1987 Dec 15;262(35):16885–16891. [PubMed] [Google Scholar]
  7. Béliveau R., Strévey J. Kinetic model for phosphate transport in renal brush-border membranes. Am J Physiol. 1988 Mar;254(3 Pt 2):F329–F336. doi: 10.1152/ajprenal.1988.254.3.F329. [DOI] [PubMed] [Google Scholar]
  8. Cheng L., Sacktor B. Sodium gradient-dependent phosphate transport in renal brush border membrane vesicles. J Biol Chem. 1981 Feb 25;256(4):1556–1564. [PubMed] [Google Scholar]
  9. DAWSON R. M. ON THE MECHANISM OF ACTION OF PHOSPHOLIPASE A. Biochem J. 1963 Sep;88:414–423. doi: 10.1042/bj0880414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Debiec H., Lorenc R. Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes. Biochem J. 1988 Oct 1;255(1):185–191. doi: 10.1042/bj2550185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gmaj P., Murer H. Cellular mechanisms of inorganic phosphate transport in kidney. Physiol Rev. 1986 Jan;66(1):36–70. doi: 10.1152/physrev.1986.66.1.36. [DOI] [PubMed] [Google Scholar]
  12. Hammerman M. R. Phosphate transport across renal proximal tubular cell membranes. Am J Physiol. 1986 Sep;251(3 Pt 2):F385–F398. doi: 10.1152/ajprenal.1986.251.3.F385. [DOI] [PubMed] [Google Scholar]
  13. Hammerman M. R., Schwab S. J. Phosphate transport in the kidney, studies with isolated membrane vesicles. Prog Clin Biol Res. 1984;168:325–330. [PubMed] [Google Scholar]
  14. Hoffmann N., Thees M., Kinne R. Phosphate transport by isolated renal brush border vesicles. Pflugers Arch. 1976 Mar 30;362(2):147–156. doi: 10.1007/BF00583641. [DOI] [PubMed] [Google Scholar]
  15. Hopfer U., Nelson K., Perrotto J., Isselbacher K. J. Glucose transport in isolated brush border membrane from rat small intestine. J Biol Chem. 1973 Jan 10;248(1):25–32. [PubMed] [Google Scholar]
  16. Kelly M. H., Hamilton J. R. A micro-technique for the assay of intestinal alkaline phosphatase. Results in normal children and in children with celiac disease. Clin Biochem. 1970 Mar;3(1):33–43. [PubMed] [Google Scholar]
  17. Kessler R. J., Vaughn D. A. Divalent metal is required for both phosphate transport and phosphate binding to phosphorin, a proteolipid isolated from brush-border membrane vesicles. J Biol Chem. 1984 Jul 25;259(14):9059–9063. [PubMed] [Google Scholar]
  18. Kessler R. J., Vaughn D. A., Fanestil D. D. Phosphate-binding proteolipid from brush border. J Biol Chem. 1982 Dec 10;257(23):14311–14317. [PubMed] [Google Scholar]
  19. Kinne R., Faust R. G. Incorporation of D-glucose-, L-alanine- and phosphate-transport systems from rat renal brush-border membranes into liposomes. Biochem J. 1977 Nov 15;168(2):311–314. doi: 10.1042/bj1680311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Koepsell H., Menuhr H., Ducis I., Wissmüller T. F. Partial purification and reconstitution of the Na+-D-glucose cotransport protein from pig renal proximal tubules. J Biol Chem. 1983 Feb 10;258(3):1888–1894. [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lin J. T., Schwarc K., Stroh A. Chromatofocussing and centrifugal reconstitution as tools for the separation and characterization of the Na+-cotransport systems of the brush-border membrane. Biochim Biophys Acta. 1984 Jul 25;774(2):254–260. doi: 10.1016/0005-2736(84)90299-2. [DOI] [PubMed] [Google Scholar]
  23. Lynch A. M., McGivan J. D. A rapid method for the reconstitution of Na+-dependent neutral amino acid transport from bovine renal brush-border membranes. Biochem J. 1987 Jun 15;244(3):503–508. doi: 10.1042/bj2440503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maloney P. C., Ambudkar S. V. Functional reconstitution of prokaryote and eukaryote membrane proteins. Arch Biochem Biophys. 1989 Feb 15;269(1):1–10. doi: 10.1016/0003-9861(89)90080-5. [DOI] [PubMed] [Google Scholar]
  25. McCormick J. I., Silvius J. R., Johnstone R. M. Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane. J Biol Chem. 1985 May 10;260(9):5706–5714. [PubMed] [Google Scholar]
  26. Mizgala C. L., Quamme G. A. Renal handling of phosphate. Physiol Rev. 1985 Apr;65(2):431–466. doi: 10.1152/physrev.1985.65.2.431. [DOI] [PubMed] [Google Scholar]
  27. Murer H., Burckhardt G. Membrane transport of anions across epithelia of mammalian small intestine and kidney proximal tubule. Rev Physiol Biochem Pharmacol. 1983;96:1–51. doi: 10.1007/BFb0031006. [DOI] [PubMed] [Google Scholar]
  28. Schäli C., Vaughn D. A., Fanestil D. D. Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding. Biochim Biophys Acta. 1984 Jan 25;769(2):277–283. doi: 10.1016/0005-2736(84)90307-9. [DOI] [PubMed] [Google Scholar]
  29. Schäli C., Vaughn D. A., Fanestil D. D. Reconstitution of the partially purified renal phosphate (Pi) transporter. Biochem J. 1986 Apr 1;235(1):189–197. doi: 10.1042/bj2350189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  31. Strévey J., Giroux S., Béliveau R. pH gradient as an additional driving force in the renal re-absorption of phosphate. Biochem J. 1990 Nov 1;271(3):687–692. doi: 10.1042/bj2710687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Szczepanska-Konkel M., Yusufi A. N., VanScoy M., Webster S. K., Dousa T. P. Phosphonocarboxylic acids as specific inhibitors of Na+-dependent transport of phosphate across renal brush border membrane. J Biol Chem. 1986 May 15;261(14):6375–6383. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES