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Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity

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Preparations of intestinal epithelial cell basal lateral plasma membranes were analyzed with free flow electrophoresis and density pertubation with digitonin. The initial basal lateral membrane preparations were obtained by equilibrium density gradient centrifugation after two different schemes of homogenization and differential sedimentation (A.K. Mircheff, C.H. van Os, and E.M. Wright. 1978.Membr. Biochem. 1: 177, and A.K. Mircheff, S.D. Hanna, M.W. Walling, and E.M. Wright. 1979.Prep. Biochem. 9:33. In these preparations, Na,K-ATPase, a marker for the basal lateral mambrane, was purified 16- to 18-fold over the initial homogenate. The preparations were also enriched in NADPH-cytochromec reductase, alkaline phosphatase, acid phosphatase, and galactosylstransferase.

Both free-flow electrophoresis, which separates on the basis of surface charge, and density perturbation with digitonin, which depends on a specific interaction of digitonin with cholesterol-rich membranes, resolved the preparation into three populations of particles. The major population, which represented basal lateral membranes purified 20- to 32-fold with respect to the initial homogenate, contained Na,K-ATPase, alkaline phosphatase, adenylate cyclase, and acid phosphatase. A second population was defined by its content of NADPH-cytochromec reductase, and the third was defined by its content of galactosyltransferase. Guanylate cyclase appeared to be partitioned between the Na,K-ATPase-rich and NADPH-cytochromec reductase-rich populations. Galactosyltransferase is also present in fractions which contain the Na,K-ATPase-rich membranes, but the present data cannot exclude the possibility of spillovers by the adjacent, galactosyltransferase-rich population. This work emphasize the importance of multiple, physical criteria for purity in the isolation of subcellular components.

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References

  1. Amar-Costesec, A., Wibo, M., Thines-Sempoux, D., Beaufay, H., Berthet, J. 1974. Analytical study of microsomes and isolated subcellular membranes from rat liver IV. Biochemical, physical, and morphological modifications of microsomal components induced by digitonin, EDTA, and phyrophosphate.J. Cell Biol. 62:717

    Google Scholar 

  2. Berezney, R., Macaulay, L.K., Crane, F.L. 1972. The purification and biochemical characterization of bovine liver nuclear membranes.J. Biol. Chem. 247:5549

    Google Scholar 

  3. Bergeron, J.J.M., Ehrenreich, J.H., Siekevitz, P., Palade, G.E. 1973. Golgi fractions prepared from rat liver homogenates. II. Biochemical characterization.J. Cell Biol. 59:73

    Google Scholar 

  4. DeDuve, C. 1974. Tissue fractionation past and present.J. Cell Biol. 50:20D

    Google Scholar 

  5. DeJonge, H.R. 1975. The localization of guanylate cyclase in rat small intestinal epithelium.Fed. Eur. Biochem. Soc. Lett. 53:237

    Google Scholar 

  6. DePierre, J.W., Karnovsky, M.L. 1973. Plasma membranes of mammalian cells. A review of methods for their characterization and isolation.J. Cell Biol. 56:275

    Google Scholar 

  7. Deppert, W., Walter, G. 1978. Cell surface glycosyltransferase — Do they exist?J. Supramol Struct. 8:19

    Google Scholar 

  8. Douglas, A.P., Kerley, R., Isselbacher, K.J. 1972. Preparation and characterization of the lateral and basal plasma membranes of the rat intestinal epithelial cell.Biochem. J. 128:1329

    Google Scholar 

  9. Eastham, E.J., Bell, J.I., Douglas, A.P. 1977. Iron-transport characteristics of vesicles of brush-border and basolateral plasma membranes from the rat enterocyte.Biochem. J. 164:289

    Google Scholar 

  10. Fleischer, B., Fleischer, S., Ozawa, H. 1969. Isolation and characterization of Golgi membranes from bovine liver.J. Cell Biol. 43:59

    Google Scholar 

  11. Fowler, S., Remacle, J., Trouet, A., Beaufay, H., Berthet, J., Wibo, M., Hauser, P. 1976. Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochromeb 5 by electron microscopy: Methodology and application to various subcellular fractions.J. Cell Biol. 71:535

    Google Scholar 

  12. Freedman, R.A., Weiser, M.M., Isselbacher, K.J. 1977. Calcium translocation by Golgi and lateral-basal membrane vesicles from rat intestine: Decrease in vitamin D-deficient rats.Proc. Nat. Acad. Sci. USA 74:3612

    Google Scholar 

  13. Fujita, M., Matsui, H., Nagano, K., Nakao, M. 1971. Asymmetric distribution of ouabain-sensitive ATPase activity in rat intestinal mucosa.Biochim. Biophys. Acta 233:404

    Google Scholar 

  14. Fujita, M., Ohta, H., Kawai, K., Matsui, H. Nakao, M. 1972. Differential isolation of microvillus and basolateral plasma membranes from intestinal mucosa: Mutually exclusive distribution of digestive enzymes and ouabain-sensitive ATPase.Biochim. Biophys. Acta 274:336

    Google Scholar 

  15. Harms, V., Hanna, S., Mircheff, A., Wright, E. 1979. The phosphatases of basal lateral membranes of rat duodenum.Fed. Proc. 38:1059

    Google Scholar 

  16. Hopfer, U., Sigrist-Nelson, K., Murer, H. 1975. Intestinal sugar transport-Studies with isolated plasma membranes.Ann. N.Y. Acad. Sci. 264:414

    Google Scholar 

  17. Howell, K.E., Ito, A., Palade, G.F. 1978. Endoplasmic reticulum marker enzymes in Golgi fractions — What does this mean?J. Cell Biol. 79:581

    Google Scholar 

  18. Ito, A., Palade, G.E. 1978. Presence of NADPH-cytochrome P-450 reductase in rat liver Golgi membranes.J. Cell Biol. 79:590

    Google Scholar 

  19. Kimura, H., Murad, F. 1975. Subcellular localization of guanylate cyclase.Life Sci. 17:837

    Google Scholar 

  20. Kimura, H., Murad, F. 1975. Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.J. Biol. Chem. 250:4810

    Google Scholar 

  21. Kinne, R., Murer, H. 1976. Polarity of epithelial cells in relation to transepithelial transport in kidney and intestine.In: Intestinal Ion Transport. J.W.L. Robinson, editor. pp. 79–95 MTP Press, Lancaster

    Google Scholar 

  22. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680

    Google Scholar 

  23. Lewis, B.A., Elkin, A., Michell, R.H., Coleman, R. 1975. Basolateral plasma membranes of intestinal epithelial cells: Identification by lactoperoxidase-catalyzed iodination and isolation after density perturbation with digitonin.Biochem. J. 152:71

    Google Scholar 

  24. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.L. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265

    Google Scholar 

  25. Mircheff, A.K. 1976. Analytical isolation of intestinal epithelial cell plasma membranes. Ph. D. dissertation. University of California, Los Angeles

    Google Scholar 

  26. Mircheff, A.K., Hanna, S.D., Walling, M.W., Wright, E. 1979. Large scale, analytical method for isolating basal lateral plasma membranes from rat duodenum.Prep. Biochem. 9(2):133

    Google Scholar 

  27. Mircheff, A.K., Van Os, C.H., Wright, E.M. 1978. Preparative scale isolation of basallateral plasma membranes from rat intestinal epithelial cells.Membrane Biochem. 1:177

    Google Scholar 

  28. Mircheff, A.K., Van Os, C.H., Wright, E.M. 1979. Alanine uptake by intestinal basallateral membrane vesicles.Fed. Proc. 38:1060

    Google Scholar 

  29. Mircheff, A.K., Walling, M.W., Van Os, C.H., Wright, E.M. 1977. Distribution of alkaline phosphatase and Ca-ATPase in intestinal epithelial cell: Differential response to 1,25(OH)2D3.In: Vitamin D. Biochemical, Chemical, and Clinical Aspects Related to Calcium Metabolism. A.W. Norman, K. Schaefer, J.W. Coburn, H.F. DeLuca, D. Fraser, H.G. Grigoleit, and D.V. Herrath, editors. pp. 281–283. Walter de Gruyter, Berlin-New York

    Google Scholar 

  30. Mircheff, A.K., Wright, E.M. 1976. Analytical isolation of plasma membranes of intestinal epithelial cells: Identification of Na,K-ATPase rich membranes and the distribution of enzyme activities.J. Membrane Biol. 28:309

    Google Scholar 

  31. Murer,H., Ammann, E., Biber, J., Hopfer, U. 1976. The surface membrane of small intestinal epithelial cells. I. Localization of adenyl cyclase.Biochim. Biophys. Acta 433:509

    Google Scholar 

  32. Murer, H., Hopfer, U., Kinne-Saffran, E., Kinne, R. 1974. Glucose transport in isolated brush border and lateral basal plasma membrane vesicle from intestinal epithelial cells.Biochim. Biophys. Acta 345:170

    Google Scholar 

  33. Podolsky, D.K., Weiser, M.M. 1975. Role of cell membrane galactosyltransferase in concanavalin A agglutination of erythrocytes.Biochem. J. 146:213

    Google Scholar 

  34. Quigley, J.P., Gotterer, G.S. 1969. Distribution of Na,K-stimulated ATPase activity in rat intestinal mucosa.Biochim. Biophys Acta 173:456

    Google Scholar 

  35. Quill, H., Weiser, M.M. 1975. Adenylate and guanylate cyclase activities and cellular differentiation in rat small intestine.Gastroenterology 69:470

    Google Scholar 

  36. Saccomani, G., Stewart, H.B., Shaw, D., Lewin, Sachs, G. 1977. Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPase containing vesicles by zonal centrifugation and free flow electrophoresis technique.Biochim. Biophys. Acta 465:311

    Google Scholar 

  37. Schachter, H., Jabbal, I., Hudgin, R.L., Pinteric, L., McGuire, E.J., Roseman, S. 1970. Intracellular localization of liver sugar nucleotide glycoprotein glycosyltransferases in a Golgi-rich fraction.J. Biol. Chem. 245:1090

    Google Scholar 

  38. Schultz, S.G., Frizzell, R.A., Nellans, H.N. 1974. Ion transports by mammalian small intestine.Annu. Rev. Physiol. 36:51

    Google Scholar 

  39. Shur, B.D., Roth, S. 1975. Cell Surface glycosyltransferases.Biochim. Biophys. Acta 415:473

    Google Scholar 

  40. Sottocasa, G.L., Kuylenstierna, B., Ernster, L., Bergstrand, A. 1967. An electron transport system in mitochondrial outer membrane.J. Cell Biol. 32:415

    Google Scholar 

  41. Stirling, C.E. 1972. Radioautographic localization of sodium pump sites in rabbit intestine.J. Cell Biol. 53:704

    Google Scholar 

  42. Thines-Sempoux, D., Amar-Costesec, A., Beaufay, H., Berthet, J. 1969. The association of cholesterol, 5′ nucleotidase, and alkaline phosphodiesterase I with a distinct group of microsomal particles.J. Cell Biol. 43:189

    Google Scholar 

  43. Van Os, C.H., Mircheff, A.K., Wright, E.M. 1977. Distribution of bicarbonate-stimulated ATPase in rat intestinal epithelium.J. Cell Biol. 73:257

    Google Scholar 

  44. Walling, M.W., Mircheff, A.K., Van Os, C.H., Wright, E.M. 1978. Subcellular distribution of nucleotide cyclases in rat intestinal epithelium.Am. J. Physiol. 235:E539

    Google Scholar 

  45. White, A.A., Karr, D.B. 1978. Improved two-step method for the assay of adenylate and guanylate cyclase.Anal. Biochem. 85:451

    Google Scholar 

  46. Wright, E.M., Van Os, C.H., Mircheff, A.K. 1979. Sugar transport across intestinal basolateral membrane vesicles.Biophys. J. 25:96a

    Google Scholar 

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Author notes

  1. A. K. Mircheff

    Present address: Department of Physiology and Biophysics, University of Southern California School of Medicine, 90033, Los Angeles, California

Authors and Affiliations

  1. Department of Physiology, University of California School of Medicine, 90024, Los Angeles, California

    A. K. Mircheff, G. Sachs, S. D. Hanna, C. S. Labiner, E. Rabon, A. P. Douglas, M. W. Walling & E. M. Wright

  2. Laboratory of Membrane Biochemistry, University of Alabama in Birmingham, 35294, Birmingham, Alabama

    A. K. Mircheff, G. Sachs, S. D. Hanna, C. S. Labiner, E. Rabon, A. P. Douglas, M. W. Walling & E. M. Wright

  3. Gastroenterology Unit, Department of Medicine, Los Angeles County-University of Southern California Medical Center, 90033, Los Angles, California

    A. K. Mircheff, G. Sachs, S. D. Hanna, C. S. Labiner, E. Rabon, A. P. Douglas, M. W. Walling & E. M. Wright

  4. Nephrology Unit, Veterans Administrations Wadsworth Hospital Center, 90024, Los Angeles, California

    A. K. Mircheff, G. Sachs, S. D. Hanna, C. S. Labiner, E. Rabon, A. P. Douglas, M. W. Walling & E. M. Wright

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Mircheff, A.K., Sachs, G., Hanna, S.D. et al. Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity. J. Membrain Biol. 50, 343–363 (1979). https://doi.org/10.1007/BF01868897

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