Summary
The [3H] phlorizin-binding component of brush border vesicles was enrichedin situ by negative purification. Several procedures, known to effect selective solubilization of membrane components, were used separately or in combination to remove proteins unrelated to the binding. Deoxycholate ruptured the vesicles and released 67% of their protein, thereby increasing the specific [3H] phlorizin-binding activity of the pellet three-to fourfold. Extracting the deoxycholate-pellets with either NaI or alkaline solutions released up to 38% of the deoxycholate-insoluble protein without significantly affecting phlorizin binding. The polypeptide composition of the membranes at the different stages was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. A number of polypeptides present in the original vesicles could be ruled out as essential components of the [3H] phlorizin binding entity.
Intact and deoxycholate-treated vesicles were subjected to proteolytic attack. Papain liberated sucrase and isomaltase from intact vesicles, but affected neither other Coomassiestained bands nor phlorizin binding. Neither the protein composition nor the binding properties of sealed vesicles were influenced by trypsin or chymotrypsin. However, all the proteolytic enzymes tested on deoxycholate-treated membranes substantially reduced [3H] phlorizin binding and produced concomitantly the disappearance of several bands from the electrophoretic profile.
Pretreatment of vesicles with papain, followed by deoxycholate extraction and incubation in alkaline media, increased the specific binding activity of the membranes up to ninefold by removing close to 90% of the protein. A limited number of polypeptides are suggested as possible candidates for the glycoside-binding site of intestinal brush borders.
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References
Alvardo, F., Crane, R. 1962. Phlorizin as a competitive inhibitor of the active transport of sugars by hamster small intestine,in vitro.Biochim. Biophys. Acta 56:170
Baldwin, S.A., Baldwin, J.M., Gorga, F.R., Lienhard, B.E. 1979. Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.Biochim. Biophys. Acta 552:183
Bode, F., Baumann, K., Diedrich, D.F. 1972. Inhibition of3H-phlorizin binding to isolated kidney brush-border membranes by phlorizin-like compounds.Biochim. Biophys. Acta 299:131
Bode, F., Baumann, K., Frasch, W., Kinne, R. 1970.Pfluegers Arch. 315:53
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72:248
Brunner, J., Hauser, H., Braun, H., Wilson, K.J., Wacker, H., O'Neill, B., Semenza, G. 1979. The mode of association of the enzyme complex sucrase-isomaltase with the intestinal brush border membrane.J. Biol. Chem. 254 (in press)
Chesney, R., Sacktor, B., Kleinzeller, A. 1974. The binding of phlorizin to the isolated luminal membrane of the renal proximal tubule.Biochim. Biophys. Acta 332:263
Cogoli, A., Eberle, A., Sigrist, H., Joss, Ch., Robinson, E., Mosimann, H., Semenza, G. 1973. Subunits of the small intestine sucrase-isomaltase complex and separation of its enzymatically active isomaltase moiety.Eur. J. Biochem. 33:40
Crane, R.K., Malathi, P., Preiser, H. 1976. Reconstitution of specific Na+-dependentd-glucose transport in liposomes by Triton X-100 extracted proteins from purified brush border membranes of rabbit kidney cortex.FEBS Lett. 67:214
Crane, R.K., Malathi, P., Preiser, H. 1976. Reconstitution of specific Na+-dependentd-glucose transport in liposomes by Triton X-100 extracted proteins from purified brush border membranes of hamster small intestine.Biochem. Biophys. Res. Commun. 71:1010
Crane, R.K., Malathi, P., Preiser, H., Fairclough, P. 1978. Some characteristics of kidney Na+-dependent glucose carrier reconstituted into sonicated liposomes.Am. J. Physiol. 234:E1
Cummins, D., Gitzelmann, R., Lindenmann, J., Semenza, G. 1968. Immunochemical study of isolated human and rabbit intestinal sucrase.Biochim. Biophys. Acta 160:396
Diedrich, D.F. 1966. Competitive inhibition of intestinal glucose transport by phlorizin analogs.Arch. Biochem. Biophys. 117:248
Frasch, W., Frohnert, P.P., Bode, F., Baumann, K., Kinne, R. 1970. Competitive inhibition of phlorizin binding byd-glucose and the influence of sodium: A study on isolated brush border membranes of kidney.Pfluegers Arch. 320:265
Furthmayr, H., Marchesi, V.T. 1976. Subunit structure of human erythrocyte glycophorin A.Biochemistry 15:1137
Glossman, H., Neville, D.J., Jr. 1972. Phlorizin receptors in isolated kidney brush border membranes.J. Biol. Chem. 247:7779
Guidotti, G. 1972. Membrane proteins.Annu. Rev. Biochem. 41:731
Kahlenberg, A. 1976. Partial purification of a membrane protein from human erythrocytes involved in glucose transport.J. Biol. Chem. 251:1582
Kalb, V.F., Jr., Bernlohr, R.W. 1977. A new spectrophotometric assay for protein in cell extracts.Anal. Biochem. 82:362
Kessler, M., Acuto, O., Storelli, C., Murer, H., Müller, M., Semenza, G. 1978. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties ofd-glucose and choline transport system.Biochim. Biophys. Acta 506:136
Kinne, R. 1976. Properties of the glucose transport system in the renal brush border membrane.Curr. Top. Membr. Trans. 8:209
Kinne, R., Faust, R.G. 1977. Incorporation ofd-glucose,l-alanine and phosphate-transport systems from rat renal brush border membranes into liposomes.Biochem. J. 168:311
Klip, A., Grinstein, S., Semenza, G. 1979. Transmembrane disposition of the phlorizin binding protein of intestinal brush borders.FEBS Lett. 99:91
Laemmli, U.K. 1970. Cleavage of structure proteins during the assembly of the head of bacteriophageT4.Nature (London) 227:680
Lane, L.C. 1978. A simple method for stabilizing protein sulfhydryl groups during SDS-gel electrophoresis.Anal. Biochem. 86:655
Lemaire, J., Maestracci, D. 1978. Labeling of a glucose binding protein in the rabbit intestinal brush border membrane.Can. J. Physiol. Pharmacol. 56:760
MacLennan, D.H. 1970. Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum.J. Biol. Chem. 245:4508
Saari, J.C. 1974. The accessibility of bovine rhodopsin in photoreceptor membranes.J. Cell Biol. 63:480
Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J., Crane, R.K. 1973. Purification of the human intestinal brush border membrane.Biochim. Biophys. Acta 323:98
Semenza, G. 1976. Small intestinal disaccharidases: Their properties and role as sugar translocators across natural and artificial membranes.In: The Enzymes of Biological Membranes. A. Matonosi, editor. Vol. 3, p. 349. Plenum Press, New York
Shanahan, M.F., Czech, M.P. 1977. Partial purification of thed-glucose transport system in rat adipocyte plasma membranes.J. Biol. Chem. 252:6554
Shanahan, M.F., Czech, M.P. 1977. Purification and reconstitution of the adipocyte plasma membraned-glucose transport system.J. Biol. Chem. 252:8341
Silverman, M. 1976. Glucose transport in the kidney.Biochim. Biophys. Acta 457:303
Singer, S.J. 1974. The molecular organization of membranes.Annu. Rev. Biochem. 43:805
Smith, M.W., Ferguson, D.R., Burton, K.A. 1975. Glucose and phlorizin protected thiol groups in pig intestinal brush border membranes.Biochem. J. 147:617
Steck, T.L. 1974. The organization of proteins in the human red cell membrane.J. Cell Biol. 62:1
Steck, T.L., Yu, J. 1977. Selective solubilization of proteins from red blood cell membranes by protein perturbants.J. Supramol. Struct. 1:220
Tannenbaum, C., Toggenburger, G., Kessler, M., Rothstein, A., Semenza, G. 1977. High-affinity phlorizin binding to brush border membranes from small intestine: Identity with (a part of) the glucose transport system, dependence on the Na+-gradient, partial purification.J. Supramol. Struct. 6:519
Thomas, L., Kinne, R. 1972. Studies on the arrangement of a glucose sensitive phlorizin binding site in the microvilli of isolated rat kidney brush border.FEBS Lett. 25:242
Tilney, L.G., Mooseker, M. 1971. Actin in the brush-border of epithelial cells of the chicken intestine.Proc. Nat. Acad. Sci. USA 68:2611
Toggenburger, G., Kessler, M., Rothstein, A., Semenza, G., Tannenbaum, C. 1978. Similarity in effects of Na+ gradients and membrane potentials ond-glucose transport by, and phlorizin binding to, vesicles derived from brush borders of rabbit intestinal mucosal cells.J. Membrane Biol. 40:269
Yu, J., Fischman, D.A., Steck, T.L. 1973. Selective solubilization of proteins and phospholipids from red blood cell membranes by non-ionic detergents.J. Supramol. Struct. 1:233
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Klip, A., Grinstein, S. & Semenza, G. Partial purification of the sugar carrier of intestinal brush border membranes. Enrichment of the phlorizin-binding component by selective extractions. J. Membrain Biol. 51, 47–73 (1979). https://doi.org/10.1007/BF01869343
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DOI: https://doi.org/10.1007/BF01869343