Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPase-containing vesicles by zonal centrifugation and free-flow electrophoresis technique

Abstract

Methods are described for purification of a vesicular membrane fraction of hog gastric mucosa using differential centrifugation, density gradient separation on zonal rotors and free-flow electrophoresis. As a result a fraction is obtained enriched 40-fold in terms of K(+)-ATPase and free of any other enzyme marker other than K(+)-activated p-nitrophenyl phosphatase. The 5'-nucleotidase and basal Mg(2+)-ATPase are clearly separated from the latter enzymes. Osmotic shock, Triton X-100 treatment or K+ ionophores increased the K(+)-ATPase activity in isotonic conditions, but K(+)-p-nitrophenyl phosphatase is not affected by these treatments, nor is the ATPase activity in the presence of NH4+. The results suggest that the electrophoretic fraction contains a major population of tight vesicles, whose permeability to K+ is rate limiting for the ATPase activity but not for the p-nitrophenyl phosphatase activity. It is concluded that K+ site for the ATPase is internal whereas the K+ site for the p-nitrophenyl phosphatase is external, hence, the K+ site must be mobile across the membrane.