Analytical Subcellular Fractionation Studies on Enterocytes from the Jejunum and Ileum of the Rat and Some Properties of Brush-Border Alkaline Phosphatase

Abstract

1. Enterocytes, isolated from the proximal jejunum and distal ileum of the rat, were homogenized and their organelles separated by isopycnic centrifugation on continuous sucrose density gradients. The distributions of marker enzymes for the principal organelles, RNA and protein were determined in the sucrose gradients and related to the activities per enterocyte. 2. In the jejunum the modal equilibrium densities of the various organelles were: brush borders (1.20), lysosomes (1.20), peroxisomes (1.19), mitochondria (1.17) and basal-lateral membranes (1.13). These values were not significantly different in the ileum. The activities of brush-border enzymes, soluble and mitochondrial malate dehydrogenase, soluble and membrane-associated lactate dehydrogenase and particulate protein content, however, were greater in the jejunal than the ileal enterocytes. 3. Detergent exposed latent alkaline phosphatase activity in jejunal enterocytes and indicated that this enzyme is present not only in the brush border but also in the basal-lateral membrane and soluble fractions of the cell. 4. Isolated jejunal brush-border preparations showed latent activities of both alkaline phosphatase and γ-glutamyltransferase whereas the activities of α-glucosidase and leucyl-β-naphthylamidase were not affected by detergent. Mechanical disruption of these preparations suggested the presence of two forms of alkaline phosphatase in the brush border and provides a technique to assess membrane fragility.