Analytical isolation of plasma membranes of intestinal epithelial cells: Identification of Na, K-ATPase rich membranes and the distribution of enzyme activities
- 1 December 1976
- journal article
- conference paper
- Published by Springer Nature in The Journal of Membrane Biology
- Vol. 28 (1) , 309-333
- https://doi.org/10.1007/bf01869703
Abstract
A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and cole material. Sucrase specific activity in the purified brush border plasma membrane was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochromec reductase, nonspecific esterase, β-glucoronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity. Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent35S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein. Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.This publication has 42 references indexed in Scilit:
- Microelectrode studies of fundic gastric mucosa: Cellular coupling and shunt conductanceThe Journal of Membrane Biology, 1974
- Passive Electrical Properties of Toad Urinary Bladder EpitheliumThe Journal of general physiology, 1974
- ION TRANSPORT BY MAMMALIAN SMALL INTESTINEAnnual Review of Physiology, 1974
- PLASMA MEMBRANES OF MAMMALIAN CELLSThe Journal of cell biology, 1973
- A test of the binary chloroplast membrane hypothesis by using a nonpenetrating chemical probe,p-(diazonium)-benzenesulfonic acidThe Journal of Membrane Biology, 1972
- The route of passive ion movement through the epithelium ofNecturus gallbladderThe Journal of Membrane Biology, 1972
- RADIOAUTOGRAPHIC LOCALIZATION OF SODIUM PUMP SITES IN RABBIT INTESTINEThe Journal of cell biology, 1972
- Surface proteins of erythrocyte membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, 1971
- AN ELECTRON-TRANSPORT SYSTEM ASSOCIATED WITH THE OUTER MEMBRANE OF LIVER MITOCHONDRIAThe Journal of cell biology, 1967
- THE COLORIMETRIC DETERMINATION OF LIPASE AND ESTERASE IN HUMAN SERUM 1Journal of Clinical Investigation, 1950