Isolation of frog urinary bladder plasma membranes with polycation coated beads

Abstract

It is now generally accepted that the increase in water permeability induced by antidiuretic hormone (ADH) in responsive epithelia is accompanied by the insertion of specific structures in the apical membrane of epithelial cells. There are strong indications that these particles, probably proteic in nature, represent water channels. In order to evaluate the nature and role of such proteins, plasma membranes were isolated by the affinity chromatography technique. The method is based on the firm attachment of the external face of the membrane to polycations covalently bound to the surface of polyacrylamide beads, followed by shearing of the rest of the cells. Maximal binding of epithelial cells to beads was achieved in a medium of low ionic strength and pH 5.2 (i.e. sucrose-MES buffer). By this procedure plasma membranes were obtained from both cAMP-stimulated cells and control cells. Membranes isolated on beads were enriched in the activity of typical membrane marker enzymes (LAP; H+ ATPase; Na+, K+ ATPase) with respect to a whole cell homogenate, whereas contamination of plasma membrane fraction by endoplasmic reticulum, lysosomes, and mitochondria was relatively low. Analysis by SDS polyacrylamide gel electrophoresis showed an interesting difference between cAMP-treated and control samples.