Complex subcellular distributions of enzymatic markers in intestinal epithelial cells

Abstract

Current procedures for isolating intestinal epithelial cell surface and intracellular membranes are based on the assumption that each organelle is marked by some unique constituent. This assumption seemed inconsistent with the dynamic picture of subcellular organization emerging from studies of membrane turnover and recycling. Therefore, we have designed an alternative fractionation which is independent of a priori marker assignments. We subjected mucosal homogenates to a sequence of separations based on sedimentation coefficient, equilibrium density, and partitioning in aqueous polymer two-phase systems. The resulting distributions of protein and enzymatic markers define a total of 17 physically and biochemically distinct membrane populations. Among these are: basal-lateral membranes, with Na,K-ATPase enriched 21-fold; brush-border membranes, with alkaline phosphatase enriched as much as 38-fold; two populations apparently derived from the endoplasmic reticulum; a series of five populations believed to have been derived from the Golgi complex; and a series of five acid phosphatase-rich populations which we cannot identify unequivocally. Each of the five enzymatic markers we have followed is associated with a multiplicity of membrane populations. Basallateral, endoplasmic reticulum, and Golgi membranes contain alkaline phosphatase at the same specific activity as the initial homogenate. Similarly, Na,K-ATPase appears to be associated with Golgi, endoplasmic reticulum, and brush-border membranes at specific activities two- to seven-fold that of the initial homogenate.