Basolateral plasma membranes of intestinal epithelial cells. Identification by lactoperoxidase catalysed iodination and isolation after density perturbation with digitonin

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  • University of Birmingham


1. Lactoperoxidase catalysed iodination was used to label intestinal epithelial cell sheets with 125 I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells and membrane bound 125 I therefore provided an effective marker for following plasma membrane fragments through subcellular fractionation procedures. 2. After homogenization and isopycnic zonal centrifugation through sucrose gradients two peaks of membrane bound 125 I were detected. One coincided with brushborder enzymes such as alkaline phosphatase, disaccharidases and L leucine β naphthylamidase, whereas the other was coincident with the major peak of (Na + + K + ) stimulated ATPase (adenosine triphosphatase), which has been thought to be concentrated in the basolateral plasma membranes of these cells. Neither peak of 125 I reflected the distribution of any marker for an intracellular organelle. 3. A large proportion of the (Na + + K + ) stimulated ATPase, and thus of the basolateral plasma membrane material, was found in a crude 'mitochondrial' fraction. It was not readily separated from mitochondria by conventional techniques of subcellular fractionation. 4. Treatment of the 'mitochondrial' fraction with digitonin increased the density of basolateral plasma membrane but had little effect on mitochondrial density. A purified preparation of digitonin loaded basolateral plasma membranes was isolated at a density of 1.20 to 1.22 by isopycnic centrifugation. 5. The enzymic composition of this preparation of basolateral plasma membranes is compared with previous preparations isolated from intestinal mucosal 'scrape' materials and from isolated cells.


Original languageEnglish
Pages (from-to)71-84
Number of pages14
JournalBiochemical Journal
Issue number1
Publication statusPublished - 1 Jan 1975