Apical membrane isolation of surface and crypt cells from rabbit distal colon
| Autor: | M.D. de Jong, C.H. van Os, R.J.M. Bindels, S. L. Abrahamse, R.J. Bridges |
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| Rok vydání: | 1996 |
| Předmět: |
Density gradient
Colon Crypt Lipid Bilayers Biophysics Regulation of salt and water reabsorption in the renal collecting duct Cell Separation Biology Cell Fractionation Biochemistry Epithelium Ion Channels H(+)-K(+)-Exchanging ATPase Animals Intestinal Mucosa Ouabain Molecular Biology GeneralLiterature_REFERENCE(e.g. dictionaries encyclopedias glossaries) Vesicle Cell Membrane Imidazoles Membrane Proteins Cell Biology Apical membrane Alkaline Phosphatase Membrane Regulatie water en zouttransport in de verzamelbuis van de nier Alkaline phosphatase Rabbits Cell fractionation Percoll |
| Zdroj: | Analytical Biochemistry, 242, 1, pp. 112-122 Analytical Biochemistry, 242, 112-122 Analytical Biochemistry, 242, pp. 112-122 |
| ISSN: | 0003-2697 |
| Popis: | Surface and crypt cells of rabbit distal colon were separately isolated, and amiloride-sensitive 22Na+ uptake could only be demonstrated in a crude membrane fraction derived from surface cells. For purification of apical membranes of surface and crypt cells (H(+)-K+)-ATPase and alkaline phosphatase were used as putative apical membrane markers. Apical membranes of surface cells were isolated after mild homogenization, low speed centrifugation, and subsequent fractionation on a Percoll density gradient. Apical membranes of crypt cells were collected after more vigorous homogenization, followed by high speed centrifugation, and fractionation on a Percoll gradient. In surface and crypt cells, (H(+)-K+)-ATPase and alkaline phosphatase activity accumulated in a low and a high density Percoll band. Further fractionation of the low density Percoll band from crypt cells on a discontinuous sucrose gradient yielded a vesicle fraction with 7- to 10-fold enrichment in (H(+)-K+)-ATPase activities. To demonstrate the usefulness of the isolated fractions in studying transport mechanisms, vesicle volume was determined and planar lipid bilayer studies were performed. In the latter studies, a 83-pS 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive Cl(-)-channel, resembling the outward rectifying intermediate conductance (ORIC) Cl(-)-channel of secretory epithelia, was encountered most frequently. This channel was present in fractions of surface and crypt cells. |
| Databáze: | OpenAIRE |
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