Purification and characterization of (H++K+)-ATPase from hog gastric mucosa.

Abstract

Abstract-A (H++K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation.The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na++K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H++K+)-ATPase and K+-stimulated p-nitrophenylphosphatase (p-NPPase).The (H++K+)-ATPase had a pH optimum of 7.4 and was stimulated by TI', K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM , respectively, at this pH. On the other hand, monovalent cations such as Na+ , Li+ and (CH3)4N+ as well as divalent cations such as Cue', Ca t+, Bat+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently.Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H++K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a major band (>90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [r-32P] ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H++K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.