Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa.
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 Tl+, 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 Cu2+, Ca2+, Ba2+, 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 (greater than or equal to 90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [gamma-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.
Hongo
T
T
Department of Membrane Biology, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Nojima
S
S
Setaka
M
M
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D001345:Autoradiography; D002413:Cations, Divalent / Q000494:pharmacology; D002498:Centrifugation; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008861:Microsomes / Q000201:enzymology; D009711:Nucleotides / Q000494:pharmacology; D010766:Phosphorylation; D013552:Swine
D002413:Cations, Divalent; D009711:Nucleotides; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase