754
BIOCHEMICAL SOCIETY TRANSACTIONS
Identification and Isolation of Basolateral Plasma Membranes from Intestinal Epithelial Cell Sheets BARBARA A. LEWIS, ALAN ELKIN, ROBERT H. MICHELL and ROGER COLEMAN Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. Several methods for the isolation of putative basolateral plasma-membrane fractions from intestinal epithelium have been described. In none of these studies, however, has unambiguous evidence been provided to demonstrate that the fractions, which were identified on the basis of their Na++K+-dependent ATPase (adenosine triphosphatase) content, originated from the basolateral plasma membranes. In order to provide rigorous identification of surface membranes, a lactoperoxidasecatalysed iodination technique was applied to a preparation of isolated epithelial cell sheets which contained a high proportion of intact viable cells. Under appropriate conditions these cells excluded lactoperoxidase, so that labelling with radioiodine was confined to the external surface of the cells. The labelled plasma membrane of these cells was then traced through subcellular fractionation by isopycnic zonal centrifugation by using the externally imposed characteristic of radioactivity as the primary marker. Two populations of iodinated plasma membranes were detected, and it was concluded that the population of labelled membranes that was separated from brushborder membranes, represented the basolateral membranes. These membranes were enriched in Na++ K+-dependent ATPase, and this enzyme was subsequently used as a marker for basolateral plasma membranes during subcellular-fractionation procedures. Isolation of basolateral plasma membranes could not be achieved by conventional techniques because of a persistent association between these membranes and mitochondria : other approaches were therefore considered. Thin&-Sempoux et al. (1 969) achieved a selective increase in density of liver plasma membranes by treatment with digitonin. We have applied this density-perturbation technique to the separation of basolateral plasma membranes from mitochondria. A crude ‘mitochondrial’ fraction was exposed to 3 mg of digitoninjmg of protein, and after isopycnic centrifugation, basolateral plasma membranes were harvested at a density of 1.20-1.22. It seems likely, as suggested by Amar-Costesec et al. (1974),that the selective increase in density was caused by the binding of digitonin to the cholesterol-rich plasma membranes. The basolateral plasma-membrane fraction, isolated by this digitonin-loading technique, appears to be of greater purity than preparations previously described. The principal contaminants in the preparation are mitochondrial inner membranes and brush-border membranes. The chief improvement over other preparations lies in the small amounts of contamination with endoplasmic-reticulum membranes. We estimate that basolateral plasma membranes constituted 60-80 % of the isolated material. This assessment of purity is based upon measurements of markers, and estimates of the relative contributions of different membranes to cell mass. The basolateral plasma-membrane fraction has been subjected to detailed chemical and enzymological analysis and its properties compared with those of the morphologically distinct brush-border membranes from the same cells. The results of these studies are presented in the preceding communication (Lewis et al., 1975). Amar-Costesec, A., Wibo, M., Thines-Sempoux, D., Beaufay, H. & Berthet, J. (1974) J. Cell Biol. 62,717-745
Lewis, B. A., Gray, G. M., Coleman, R. & Michell, R. H. (1975) Biochem. SOC.Trans.3,752-753 Thinks-Sempoux, D., her-Costesec, A., Beaufay, H. & Berthet, J. (1969) J. Cell. Biol. 43, 189-1 92
1975
BIOCHEMICAL SOCIETY TRANSACTIONS
Identification and Isolation of Basolateral Plasma Membranes from Intestinal Epithelial Cell Sheets BARBARA A. LEWIS, ALAN ELKIN, ROBERT H. MICHELL and ROGER COLEMAN Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. Several methods for the isolation of putative basolateral plasma-membrane fractions from intestinal epithelium have been described. In none of these studies, however, has unambiguous evidence been provided to demonstrate that the fractions, which were identified on the basis of their Na++K+-dependent ATPase (adenosine triphosphatase) content, originated from the basolateral plasma membranes. In order to provide rigorous identification of surface membranes, a lactoperoxidasecatalysed iodination technique was applied to a preparation of isolated epithelial cell sheets which contained a high proportion of intact viable cells. Under appropriate conditions these cells excluded lactoperoxidase, so that labelling with radioiodine was confined to the external surface of the cells. The labelled plasma membrane of these cells was then traced through subcellular fractionation by isopycnic zonal centrifugation by using the externally imposed characteristic of radioactivity as the primary marker. Two populations of iodinated plasma membranes were detected, and it was concluded that the population of labelled membranes that was separated from brushborder membranes, represented the basolateral membranes. These membranes were enriched in Na++ K+-dependent ATPase, and this enzyme was subsequently used as a marker for basolateral plasma membranes during subcellular-fractionation procedures. Isolation of basolateral plasma membranes could not be achieved by conventional techniques because of a persistent association between these membranes and mitochondria : other approaches were therefore considered. Thin&-Sempoux et al. (1 969) achieved a selective increase in density of liver plasma membranes by treatment with digitonin. We have applied this density-perturbation technique to the separation of basolateral plasma membranes from mitochondria. A crude ‘mitochondrial’ fraction was exposed to 3 mg of digitoninjmg of protein, and after isopycnic centrifugation, basolateral plasma membranes were harvested at a density of 1.20-1.22. It seems likely, as suggested by Amar-Costesec et al. (1974),that the selective increase in density was caused by the binding of digitonin to the cholesterol-rich plasma membranes. The basolateral plasma-membrane fraction, isolated by this digitonin-loading technique, appears to be of greater purity than preparations previously described. The principal contaminants in the preparation are mitochondrial inner membranes and brush-border membranes. The chief improvement over other preparations lies in the small amounts of contamination with endoplasmic-reticulum membranes. We estimate that basolateral plasma membranes constituted 60-80 % of the isolated material. This assessment of purity is based upon measurements of markers, and estimates of the relative contributions of different membranes to cell mass. The basolateral plasma-membrane fraction has been subjected to detailed chemical and enzymological analysis and its properties compared with those of the morphologically distinct brush-border membranes from the same cells. The results of these studies are presented in the preceding communication (Lewis et al., 1975). Amar-Costesec, A., Wibo, M., Thines-Sempoux, D., Beaufay, H. & Berthet, J. (1974) J. Cell Biol. 62,717-745
Lewis, B. A., Gray, G. M., Coleman, R. & Michell, R. H. (1975) Biochem. SOC.Trans.3,752-753 Thinks-Sempoux, D., her-Costesec, A., Beaufay, H. & Berthet, J. (1969) J. Cell. Biol. 43, 189-1 92
1975
