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. 1982 Nov 15;208(2):377–382. doi: 10.1042/bj2080377

An efficient method for the isolation and separation of basolateral-membrane and luminal-membrane vesicles from rabbit kidney cortex.

M I Sheikh, U Kragh-Hansen, K E Jørgensen, H Røigaard-Petersen
PMCID: PMC1153973  PMID: 7159406

Abstract

A procedure for isolation and separation of purified luminal-membrane and basolateral-membrane vesicles from adult and newborn rabbit renal cortex by using Ca2+/Mg2+ precipitation, differential centrifugation and a self-orienting Percoll-gradient centrifugation is described. The purity of the membrane-vesicle suspensions was examined by electron microscopy and by measuring the activity of several marker enzymes. The activity of Na+ + K+-stimulated ATPase in the fraction mainly containing adult rabbit basolateral-membrane vesicles was enriched 16-fold, and the activity of alkaline phosphatase in the fraction mainly containing luminal-membrane vesicles was increased 13-fold, compared with the homogenate. Similar results were obtained with kidneys from newborn rabbits. Uptake studies, with a rapid filtration technique and the spectrophotometric method described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J. 208, 359-368], showed that both adult and newborn rabbit luminal-membrane vesicles, in contrast with the basolateral-membrane preparations, possess an Na+-dependent electrogenic transport system for L-proline. Adult rabbit luminal-membrane vesicles take up citrate and L-malate by Na+-dependent electrogenic processes, whereas adult rabbit basolateral membrane vesicles do not exhibit electrogenic uptake of citrate. By contrast, these vesicles show Na+-dependent electrogenic uptake of L-malate.

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Selected References

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

  1. Baerlocher K. E., Scriver C. R., Mohyuddin F. The ontogeny of amino acid transport in rat kidney. I. Effect on distribution ratios and intracellular metabolism of proline and glycine. Biochim Biophys Acta. 1971 Dec 3;249(2):353–363. doi: 10.1016/0005-2736(71)90114-3. [DOI] [PubMed] [Google Scholar]
  2. Baerlocher K. E., Scriver C. R., Mohyuddin F. The ontogeny of amino acid transport in rat kidney. II. Kinetics of uptake and effect of anoxia. Biochim Biophys Acta. 1971 Dec 3;249(2):364–372. doi: 10.1016/0005-2736(71)90115-5. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Brodehl J., Gellissen K. Endogenous renal transport of free amino acids in infancy and childhood. Pediatrics. 1968 Sep;42(3):395–404. [PubMed] [Google Scholar]
  5. CROFT D. N., LUBRAN M. THE ESTIMATION OF DEOXYRIBONUCLEIC ACID IN THE PRESENCE OF SIALIC ACID: APPLICATION TO ANALYSIS OF HUMAN GASTRIC WASHINGS. Biochem J. 1965 Jun;95:612–620. doi: 10.1042/bj0950612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crane R. K., Malathi P., Preiser H. Reconstitution of specific Na+-dependent D-glucose transport in liposomes by Triton X-100-extracted proteins from purified brush border membranes of rabbit kidney cortex. FEBS Lett. 1976 Aug 15;67(2):214–216. doi: 10.1016/0014-5793(76)80369-9. [DOI] [PubMed] [Google Scholar]
  7. Goldmann D. R., Roth K. S., Langfitt T. W., Jr, Segal S. L-proline transport by newborn rat kidney brush-border membrane vesicles. Biochem J. 1979 Jan 15;178(1):253–256. doi: 10.1042/bj1780253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. HUEBSCHER G., WEST G. R. SPECIFIC ASSAYS OF SOME PHOSPHATASES IN SUBCELLULAR FRACTIONS OF SMALL INTESTINAL MUCOSA. Nature. 1965 Feb 20;205:799–800. doi: 10.1038/205799a0. [DOI] [PubMed] [Google Scholar]
  9. Heidrich H. G., Kinne R., Kinne-Saffran E., Hannig K. The polarity of the proximal tubule cell in rat kidney. Different surface charges for the brush-border microvilli and plasma membranes from the basal infoldings. J Cell Biol. 1972 Aug;54(2):232–245. doi: 10.1083/jcb.54.2.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jørgensen P. L. Sodium and potassium ion pump in kidney tubules. Physiol Rev. 1980 Jul;60(3):864–917. doi: 10.1152/physrev.1980.60.3.864. [DOI] [PubMed] [Google Scholar]
  11. Kragh-Hansen U., Jørgensen K. E., Sheikh M. I. The use of a potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Uptake of L-malate and D-malate by luminal-membrane vesicles. Biochem J. 1982 Nov 15;208(2):369–376. doi: 10.1042/bj2080369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kragh-Hansen U., Jørgensen K. E., Sheikh M. I. The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements. Biochem J. 1982 Nov 15;208(2):359–368. doi: 10.1042/bj2080359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Murer H., Kinne R. The use of isolated membrane vesicles to study epithelial transport processes. J Membr Biol. 1980 Jul 15;55(2):81–95. doi: 10.1007/BF01871151. [DOI] [PubMed] [Google Scholar]
  14. Ottolenghi P. The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. Biochem J. 1975 Oct;151(1):61–66. doi: 10.1042/bj1510061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  16. Reynolds R. A., Wald H., McNamara P. D., Segal S. An improved method for isolation of basolateral membranes from rat kidney. Biochim Biophys Acta. 1980 Sep 2;601(1):92–100. doi: 10.1016/0005-2736(80)90516-7. [DOI] [PubMed] [Google Scholar]
  17. Rostgaard J., Moller O. J. Electron microscopy of a microsomal fraction ritch in (Na++K+)-ATPase and isolated from kidney cortex. Structural changes accompanying freeze- and DOC-activation and identification of main components. Exp Cell Res. 1971 Oct;68(2):356–371. doi: 10.1016/0014-4827(71)90161-3. [DOI] [PubMed] [Google Scholar]
  18. Sacktor B. Trehalase and the transport of glucose in the mammalian kidney and intestine. Proc Natl Acad Sci U S A. 1968 Jul;60(3):1007–1014. doi: 10.1073/pnas.60.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scalera V., Huang Y. K., Hildmann B., Murer H. A simple isolation method for basal-lateral plasma membranes from rat kidney cortex. Membr Biochem. 1981;4(1):49–61. doi: 10.3109/09687688109065422. [DOI] [PubMed] [Google Scholar]
  20. Sheikh M. I., Møller J. V. Renal handling of phenol red. IV. Tubular localization in rabbit and rat kidney in vivo. Am J Physiol. 1980 Mar;238(3):F159–F165. doi: 10.1152/ajprenal.1980.238.3.F159. [DOI] [PubMed] [Google Scholar]
  21. Sheikh M. I. Renal handling of phenol red. I. A comparative study on the accumulation of phenol red and p-aminohippurate in rabbit kidney tubules in vitro. J Physiol. 1972 Dec;227(2):565–590. doi: 10.1113/jphysiol.1972.sp010048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wright S. H., Krasne S., Kippen I., Wright E. M. Na+-dependent transport of tricarboxylic acid cycle intermediates by renal brush border membranes. Effects on fluorescence of a potential-sensitive cyanine dye. Biochim Biophys Acta. 1981 Feb 6;640(3):767–778. doi: 10.1016/0005-2736(81)90107-3. [DOI] [PubMed] [Google Scholar]

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