A vesicular preparation enriched in basolateral membranes from cow ciliary body epithelium

Abstract

A procedure is described for the preparation of a tissue fraction enriched in basal-lateral membranes of the epithelium of ciliary body dissected from abattoir-derived cow eyes. Microscopical methods established the viability of the epithelial cells in the dissected tissue. A two-step homogenization procedure was employed. First, mild trituration with a Potter-Elvehjem tissue grinder was used to release the epithelial cells with minimal disintegration of the stroma and muscle components. Then, complete vesicula-tion of the released material was induced with a Dounce homogenizer. This material was fractionated through sequential differential and (Ficoll 400) equilibrium density gradient centrifugation steps. The procedure led to the isolation of a fraction enriched 16.8 ± 5.5 (± SE, n-5) fold in Na +K⁺-ATPase activity with respect to the initial homogenate. Using a method to artificially generate pH gradients it was demonstrated that the preparation contained sealed vesicles and that the membranes of these vesicles exhibited a K⁺ selectivity consistent with a plasma membrane origin.