Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPase-containing vesicles by zonal centrifugation and free-flow electrophoresis technique.
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.
Saccomani
G
G
Laboratory of Membrane Biology, University of Alabama in Birmingham, Birmingham, Ala 35294, USA.
Stewart
H B
HB
Shaw
D
D
Lewin
M
M
Sachs
G
G
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D005753:Gastric Mucosa / Q000378:metabolism* / Q000473:pathology; D008274:Magnesium / Q000737:chemistry; D008854:Microscopy, Electron; D017830:Octoxynol / Q000494:pharmacology; D009995:Osmosis; D009997:Osmotic Pressure; D010455:Peptides / Q000737:chemistry; D012413:Rubidium / Q000494:pharmacology; D013552:Swine; D014634:Valinomycin / Q000494:pharmacology
D027682:Cation Transport Proteins; D010455:Peptides; D014634:Valinomycin; D000255:Adenosine Triphosphate; D017830:Octoxynol; D009597:4-Nitrophenylphosphatase; D000251:Adenosine Triphosphatases; C022761:potassium transporting ATPase; D008274:Magnesium; D012413:Rubidium