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

Abstract

1. Lactoperoxidase-catalysed iodination was used to label intestinal epithelial cell sheets with ¹²⁵I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells and membrane-bound ¹²⁵I 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 ¹²⁵I were detected. One coincided with brush border 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 125I reflected the distribution of any marker for an intracellular organelle. 3. A larger 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 readiily 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 1.20-1.22 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.