Membrane populations of bovine choroid plexus: separation by density gradient centrifugation in modified colloidal silica.

Abstract

Abstract: The choroid plexus is intimately involved in the production and regulation of the cerebrospinal fluid. Populations of surface membranes from this epithelial tissue were separated by density gradient centrifugation by use of modified colloidal silica (Percoll). A fraction of heavy microsomes (P3) containing plasma membranes was prepared by differential centrifugation. Membranes in fraction P3 were mixed with a given concentration of Percoll and density gradients generated during centrifugation. When fraction P3 was mixed with 20% (v/v) Percoll and centrifuged at 20,000 r.p.m. for 1 h in a 50.2 Ti fixed‐angle rotor, membranes containing alkaline phosphatase (AP) were found at a density of 1.037 g/cm3 while those containing NaK ATPase were found at 1.047 g/cm3. With more shallow density gradients using 12% and 14% Percoll, a broad shoulder of AP activity became manifest at densities greater than 1.060 g/cm3 suggesting multiple populations of membranes containing AP. Membranes containing AP could also be separated from membranes containing γ‐glutamyl transpeptidase (γ‐GTP); this separation was most pronounced in 12% Percoll. The activity of γ‐GTP could not be separated from activity of NaK ATPase. Total protein was distributed broadly throughout the gradients. Studies have been undertaken to compare the behavior of choroidal membranes in Percoll gradients with that of renal membranes because the biochemical anatomy of the kidney has been extensively studied. In contrast to choroidal membranes, renal membranes with NaK ATPase activity were found to have densities lower than those membranes with AP. Thus, the distribution of membrane‐bound enzymes from kidney in a Percoll gradient was exactly the opposite of that observed for these same enzymes from choroid plexus. In addition, unlike the γ‐GTP activity of choroid plexus, γ‐GTP from kidney could be separated from the activities of both alkaline phosphatase and NaK ATPase. These marked differences in membrane populations between choroid plexus and kidney as defined by Percoll density gradient centrifugation analyses are presumably reflective of differences in the functions of the two epithelial tissues.