Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity

Abstract

Preparations of intestinal epithelial cell basal lateral plasma membranes were analyzed with free flow electrophoresis and density perturbation with digitonin. The initial basal lateral membrane preparations were obtained by equilibrium density gradient centrifugation after two different schemes of homogenization and differential sedimentation (A.K. Mircheff, C.H. van Os, and E.M. Wright. 1978. Membr. Biochem. 1:177, and A.K. Mircheff, S.D. Hanna, M.W. Walling, and E.M. Wright. 1979. Prep. Biochem. 9:33. In these preparations, Na,K-ATPase, a marker for the basal lateral mambrane, was purified 16- to 18-fold over the initial homogenate. The preparations were also enriched in NADPH-cytochrome c reductase, alkaline phosphatase, acid phosphatase, and galactosyltransferase. Both free-flow electrophoresis, which separates on the basis of surface charge, and density perturbation with digitonin, which depends on a specific interaction of digitonin with cholesterol-rich membranes, resolved the preparation into three populations of particles. The major population, which represented basal lateral membranes purified 20- to 32-fold with respect to the initial homogenate, contained Na,K-ATPase, alkaline phosphatase, adenylate cyclase, and acid phosphatase. A second population was defined by its content of NADPH-cytochrome c reductase, and the third was defined by its content of galactosyltransferase. Guanylate cyclase appeared to be partitioned between the Na,K-ATPase-rich and NADPH-cytochrome c reductase-rich populations. Galactosyltransferase is also present in fractions which contain the Na,K-ATPase-rich membranes, but the present data cannot exclude the possibility of spillover by the adjacent, galactosyltransferase-rich population. This work emphasizes the importance of multiple, physical criteria for purity in the isolation of subcellular components.