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Purification and characterization of (H+ + K+)-ATPase from hog gastric mucosa
1Department of Membrane Biology, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
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 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.
Insights
Researchers developed a simple method to isolate highly active gastric (H+ + K+)-ATPase enzyme fractions from hog stomach membranes. This method yields a purified enzyme crucial for understanding stomach acid secretion and developing related therapies.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- The gastric (H+ + K+)-ATPase is a key enzyme responsible for acid secretion in the stomach.
- Efficient isolation of this enzyme is crucial for studying its function and developing therapeutic interventions.
Purpose of the Study:
- To develop a simple and effective method for isolating a highly purified and active (H+ + K+)-ATPase-enriched membrane fraction.
- To characterize the enzymatic properties of the isolated (H+ + K+)-ATPase.
Main Methods:
- Differential and repeated 7% Ficoll gradient centrifugation of hog gastric mucosa.
- Enzyme activity assays for (H+ + K+)-ATPase and other markers.
- Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) for protein analysis.
- Enzyme kinetics studies including pH optimum and cation effects.
Main Results:
- A microsomal membrane fraction highly enriched in (H+ + K+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (p-NPPase) was obtained.
- The isolated enzyme exhibited optimal activity at pH 7.4 and was stimulated by Tl+, K+, Rb+, and NH4+.
- Omeprazole, a specific inhibitor, demonstrated pH-dependent inhibition, while other cations and inhibitors showed no effect or concentration-dependent inhibition.
- SDS-PAGE revealed a major protein band at 97,400 daltons, identified as the enzyme's alpha-subunit, which underwent phosphorylation and dephosphorylation.
Conclusions:
- The described Ficoll gradient centrifugation method is a simple and highly effective technique for isolating active gastric (H+ + K+)-ATPase.
- The characterized enzyme properties provide valuable insights into its function in gastric acid secretion.
- This purified enzyme fraction serves as a robust model for further biochemical and pharmacological studies.