Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Sep 1;109(3):1057–1069. doi: 10.1083/jcb.109.3.1057

(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit

PMCID: PMC2115743  PMID: 2549076

Abstract

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

Full Text

The Full Text of this article is available as a PDF (4.5 MB).

Selected References

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

  1. Almers W., Stirling C. Distribution of transport proteins over animal cell membranes. J Membr Biol. 1984;77(3):169–186. doi: 10.1007/BF01870567. [DOI] [PubMed] [Google Scholar]
  2. Ames G. F., Nikaido K. Two-dimensional gel electrophoresis of membrane proteins. Biochemistry. 1976 Feb 10;15(3):616–623. doi: 10.1021/bi00648a026. [DOI] [PubMed] [Google Scholar]
  3. Bonkovsky H. L., Hauri H. P., Marti U., Gasser R., Meyer U. A. Cytochrome P450 of small intestinal epithelial cells. Immunochemical characterization of the increase in cytochrome P450 caused by phenobarbital. Gastroenterology. 1985 Feb;88(2):458–467. doi: 10.1016/0016-5085(85)90507-4. [DOI] [PubMed] [Google Scholar]
  4. Brown T. A., Horowitz B., Miller R. P., McDonough A. A., Farley R. A. Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney. Biochim Biophys Acta. 1987 Apr 8;912(2):244–253. doi: 10.1016/0167-4838(87)90095-1. [DOI] [PubMed] [Google Scholar]
  5. Browning T. H., Trier J. S. Organ culture of mucosal biopsies of human small intestine. J Clin Invest. 1969 Aug;48(8):1423–1432. doi: 10.1172/JCI106108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caplan M. J., Anderson H. C., Palade G. E., Jamieson J. D. Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes. Cell. 1986 Aug 15;46(4):623–631. doi: 10.1016/0092-8674(86)90888-3. [DOI] [PubMed] [Google Scholar]
  7. Caplan M. J., Palade G. E., Jamieson J. D. Newly synthesized Na,K-ATPase alpha-subunit has no cytosolic intermediate in MDCK cells. J Biol Chem. 1986 Feb 25;261(6):2860–2865. [PubMed] [Google Scholar]
  8. Chehab F. F., Kan Y. W., Law M. L., Hartz J., Kao F. T., Blostein R. Human placental Na+,K+-ATPase alpha subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7901–7905. doi: 10.1073/pnas.84.22.7901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  10. Conteas C. N., McDonough A. A., Kozlowski T. R., Hensley C. B., Wood R. L., Mircheff A. K. Mapping subcellular distribution of Na+-K+-ATPase in rat parotid gland. Am J Physiol. 1986 Mar;250(3 Pt 1):C430–C441. doi: 10.1152/ajpcell.1986.250.3.C430. [DOI] [PubMed] [Google Scholar]
  11. Coudrier E., Reggio H., Louvard D. Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli. EMBO J. 1983;2(3):469–475. doi: 10.1002/j.1460-2075.1983.tb01446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dottavio-Martin D., Ravel J. M. Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride. Anal Biochem. 1978 Jul 1;87(2):562–565. doi: 10.1016/0003-2697(78)90706-6. [DOI] [PubMed] [Google Scholar]
  13. Ernst S. A., Schreiber J. H. Ultrastructural localization of Na+,K+-ATPase in rat and rabbit kidney medulla. J Cell Biol. 1981 Dec;91(3 Pt 1):803–813. doi: 10.1083/jcb.91.3.803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Esmann M., Skou J. C., Christiansen C. Solubilization and molecular weight determination of the (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. Biochim Biophys Acta. 1979 Apr 12;567(2):410–420. doi: 10.1016/0005-2744(79)90127-x. [DOI] [PubMed] [Google Scholar]
  15. Fambrough D. M., Bayne E. K. Multiple forms of (Na+ + K+)-ATPase in the chicken. Selective detection of the major nerve, skeletal muscle, and kidney form by a monoclonal antibody. J Biol Chem. 1983 Mar 25;258(6):3926–3935. [PubMed] [Google Scholar]
  16. Geering K., Meyer D. I., Paccolat M. P., Kraehenbühl J. P., Rossier B. C. Membrane insertion of alpha- and beta-subunits of Na+,K+-ATPase. J Biol Chem. 1985 Apr 25;260(8):5154–5160. [PubMed] [Google Scholar]
  17. Gerard C., Gabrion J., Verrier B., Reggio H., Mauchamp J. Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells. Eur J Cell Biol. 1985 Jul;38(1):134–141. [PubMed] [Google Scholar]
  18. Girardet M., Geering K., Rossier B. C., Kraehenbuhl J. P., Bron C. Hydrophobic labeling of (Na+,K+)-ATPase: further evidence that the beta subunit is embedded in the membrane bilayer. Biochemistry. 1983 Apr 26;22(9):2296–2300. doi: 10.1021/bi00278a037. [DOI] [PubMed] [Google Scholar]
  19. Gorr S. U., Stieger B., Fransen J. A., Kedinger M., Marxer A., Hauri H. P. A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells. J Cell Biol. 1988 Jun;106(6):1937–1946. doi: 10.1083/jcb.106.6.1937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gorvel J. P., Liabeuf A., Massey D., Liot D., Goridis C., Maroux S. Recognition of sodium- and potassium-dependent adenosine triphosphatase in organs of the mouse by means of a monoclonal antibody. Cell Tissue Res. 1983;234(3):619–632. doi: 10.1007/BF00218655. [DOI] [PubMed] [Google Scholar]
  21. Gustin M. C., Goodman D. B. Characterization of the phosphorylated intermediate of the K+-ouabain-insensitive ATPase of the rabbit colon brush-border membrane. J Biol Chem. 1982 Aug 25;257(16):9629–9633. [PubMed] [Google Scholar]
  22. Gustin M. C., Goodman D. B. Isolation of brush-border membrane from the rabbit descending colon epithelium. Partial characterization of a unique K+-activated ATPase. J Biol Chem. 1981 Oct 25;256(20):10651–10656. [PubMed] [Google Scholar]
  23. Hagenbuch B., Stange G., Murer H. Transport of sulphate in rat jejunal and rat proximal tubular basolateral membrane vesicles. Pflugers Arch. 1985 Oct;405(3):202–208. doi: 10.1007/BF00582561. [DOI] [PubMed] [Google Scholar]
  24. Hauri H. P., Bucher K. Immunoblotting with monoclonal antibodies: importance of the blocking solution. Anal Biochem. 1986 Dec;159(2):386–389. doi: 10.1016/0003-2697(86)90357-x. [DOI] [PubMed] [Google Scholar]
  25. Hauri H. P., Kedinger M., Haffen K., Grenier J. F., Hadorn B. Organ culture of human duodenum and jejunum. Biol Gastroenterol (Paris) 1975 Oct-Nov;8(4):307–319. [PubMed] [Google Scholar]
  26. Hauri H. P., Sterchi E. E., Bienz D., Fransen J. A., Marxer A. Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. J Cell Biol. 1985 Sep;101(3):838–851. doi: 10.1083/jcb.101.3.838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hiatt A., McDonough A. A., Edelman I. S. Assembly of the (Na+ + K+)-adenosine triphosphatase. Post-translational membrane integration of the alpha subunit. J Biol Chem. 1984 Feb 25;259(4):2629–2635. [PubMed] [Google Scholar]
  28. Jørgensen P. L. Structure, function and regulation of Na,K-ATPase in the kidney. Kidney Int. 1986 Jan;29(1):10–20. doi: 10.1038/ki.1986.3. [DOI] [PubMed] [Google Scholar]
  29. Kashgarian M., Biemesderfer D., Caplan M., Forbush B., 3rd Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. Kidney Int. 1985 Dec;28(6):899–913. doi: 10.1038/ki.1985.216. [DOI] [PubMed] [Google Scholar]
  30. Kawakami K., Noguchi S., Noda M., Takahashi H., Ohta T., Kawamura M., Nojima H., Nagano K., Hirose T., Inayama S. Primary structure of the alpha-subunit of Torpedo californica (Na+ + K+)ATPase deduced from cDNA sequence. Nature. 1985 Aug 22;316(6030):733–736. doi: 10.1038/316733a0. [DOI] [PubMed] [Google Scholar]
  31. Kyte J. Immunoferritin determination of the distribution of (Na+ + K+) ATPase over the plasma membranes of renal convoluted tubules. I. Distal segment. J Cell Biol. 1976 Feb;68(2):287–303. doi: 10.1083/jcb.68.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kyte J. Immunoferritin determination of the distribution of (Na+ + K+) ATPase over the plasma membranes of renal convoluted tubules. II. Proximal segment. J Cell Biol. 1976 Feb;68(2):304–318. doi: 10.1083/jcb.68.2.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Louvard D. Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4132–4136. doi: 10.1073/pnas.77.7.4132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Low M. G., Zilversmit D. B. Role of phosphatidylinositol in attachment of alkaline phosphatase to membranes. Biochemistry. 1980 Aug 19;19(17):3913–3918. doi: 10.1021/bi00558a004. [DOI] [PubMed] [Google Scholar]
  36. Marshall P. J., Hokin L. E. Microheterogeneity of the glycoprotein subunit of the (sodium + potassium)-activated adenosine triphosphatase from the electroplax of Electrophorus electricus. Biochem Biophys Res Commun. 1979 Mar 30;87(2):476–482. doi: 10.1016/0006-291x(79)91820-5. [DOI] [PubMed] [Google Scholar]
  37. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  38. Noguchi S., Mishina M., Kawamura M., Numa S. Expression of functional (Na+ + K+)-ATPase from cloned cDNAs. FEBS Lett. 1987 Dec 10;225(1-2):27–32. doi: 10.1016/0014-5793(87)81125-0. [DOI] [PubMed] [Google Scholar]
  39. Noguchi S., Noda M., Takahashi H., Kawakami K., Ohta T., Nagano K., Hirose T., Inayama S., Kawamura M., Numa S. Primary structure of the beta-subunit of Torpedo californica (Na+ + K+)-ATPase deduced from the cDNA sequence. FEBS Lett. 1986 Feb 17;196(2):315–320. doi: 10.1016/0014-5793(86)80270-8. [DOI] [PubMed] [Google Scholar]
  40. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  41. Ovchinnikov YuA, Arystarkhova E. A., Arzamazova N. M., Dzhandzhugazyan K. N., Efremov R. G., Nabiev I. R., Modyanov N. N. Differentiated analysis of the secondary structure of hydrophilic and hydrophobic regions in alpha- and beta-subunits of Na+,K+-ATPase by Raman spectroscopy. FEBS Lett. 1988 Jan 25;227(2):235–239. doi: 10.1016/0014-5793(88)80905-0. [DOI] [PubMed] [Google Scholar]
  42. Ovchinnikov YuA, Modyanov N. N., Broude N. E., Petrukhin K. E., Grishin A. V., Arzamazova N. M., Aldanova N. A., Monastyrskaya G. S., Sverdlov E. D. Pig kidney Na+,K+-ATPase. Primary structure and spatial organization. FEBS Lett. 1986 Jun 9;201(2):237–245. doi: 10.1016/0014-5793(86)80616-0. [DOI] [PubMed] [Google Scholar]
  43. Quaroni A., Isselbacher K. J. Cytotoxic effects and metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in duodenal and ileal epithelial cell cultures. J Natl Cancer Inst. 1981 Dec;67(6):1353–1362. [PubMed] [Google Scholar]
  44. Quaroni A., Isselbacher K. J. Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface components. Dev Biol. 1985 Oct;111(2):267–279. doi: 10.1016/0012-1606(85)90482-8. [DOI] [PubMed] [Google Scholar]
  45. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schenk D. B., Leffert H. L. Monoclonal antibodies to rat Na+,K+-ATPase block enzymatic activity. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5281–5285. doi: 10.1073/pnas.80.17.5281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schultz S. G. A cellular model for active sodium absorption by mammalian colon. Annu Rev Physiol. 1984;46:435–451. doi: 10.1146/annurev.ph.46.030184.002251. [DOI] [PubMed] [Google Scholar]
  48. Shull G. E., Lane L. K., Lingrel J. B. Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA. Nature. 1986 May 22;321(6068):429–431. doi: 10.1038/321429a0. [DOI] [PubMed] [Google Scholar]
  49. Shull G. E., Schwartz A., Lingrel J. B. Amino-acid sequence of the catalytic subunit of the (Na+ + K+)ATPase deduced from a complementary DNA. Nature. 1985 Aug 22;316(6030):691–695. doi: 10.1038/316691a0. [DOI] [PubMed] [Google Scholar]
  50. Stieger B., Marxer A., Hauri H. P. Isolation of brush-border membranes from rat and rabbit colonocytes: is alkaline phosphatase a marker enzyme? J Membr Biol. 1986;91(1):19–31. doi: 10.1007/BF01870211. [DOI] [PubMed] [Google Scholar]
  51. Takemura S., Omori K., Tanaka K., Omori K., Matsuura S., Tashiro Y. Quantitative immunoferritin localization of [Na+,K+]ATPase on canine hepatocyte cell surface. J Cell Biol. 1984 Oct;99(4 Pt 1):1502–1510. doi: 10.1083/jcb.99.4.1502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tamkun M. M., Fambrough D. M. The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport. J Biol Chem. 1986 Jan 25;261(3):1009–1019. [PubMed] [Google Scholar]
  53. Thompson J. A., Lau A. L., Cunningham D. D. Selective radiolabeling of cell surface proteins to a high specific activity. Biochemistry. 1987 Feb 10;26(3):743–750. doi: 10.1021/bi00377a014. [DOI] [PubMed] [Google Scholar]
  54. Tokuyasu K. T. A study of positive staining of ultrathin frozen sections. J Ultrastruct Res. 1978 Jun;63(3):287–307. doi: 10.1016/s0022-5320(78)80053-7. [DOI] [PubMed] [Google Scholar]
  55. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weiser M. M. Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation. J Biol Chem. 1973 Apr 10;248(7):2536–2541. [PubMed] [Google Scholar]
  57. Weiser M. M., Neumeier M. M., Quaroni A., Kirsch K. Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane. J Cell Biol. 1978 Jun;77(3):722–734. doi: 10.1083/jcb.77.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES