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Solubilization of active (H+ + K+)-ATPase from gastric membrane

A Soumarmon, F Grelac, M J Lewin

    Biochimica Et Biophysica Acta
    |August 10, 1983
    PubMed

    Insights

    Researchers successfully solubilized the gastric (H+ + K+)-ATPase enzyme using n-octylglucoside. The extracted enzyme retained its characteristic K+-stimulated activity and phosphorylation properties, indicating successful isolation of an active proton pump.

    Area of Science:

    • Biochemistry
    • Membrane Protein Biochemistry
    • Enzymology

    Background:

    • The gastric proton pump, (H+ + K+)-ATPase, plays a crucial role in acid secretion.
    • Understanding its structure and function requires methods for isolating the active enzyme.
    • Previous attempts to solubilize (H+ + K+)-ATPase have faced challenges in retaining enzyme activity.

    Purpose of the Study:

    • To develop a method for solubilizing the gastric (H+ + K+)-ATPase while preserving its enzymatic activity.
    • To characterize the biochemical properties of the solubilized (H+ + K+)-ATPase.
    • To provide the first evidence of a successfully solubilized and active gastric proton pump.

    Main Methods:

    • Preparation of (H+ + K+)-ATPase-enriched membranes from hog gastric mucosa using sucrose gradient centrifugation.
    • Solubilization of membrane proteins using 1-2% n-octylglucoside.
    • Characterization of enzyme activity (Mg2+-ATPase, p-nitrophenylphosphatase) and phosphorylation in soluble and precipitated fractions.
    • Analysis of molecular weight using glycerol gradient centrifugation.

    Main Results:

    • Solubilized (H+ + K+)-ATPase retained K+-stimulated Mg2+-ATPase and p-nitrophenylphosphatase activities.
    • The enzyme exhibited K+-sensitive phosphorylation and dephosphorylation.
    • Glycerol gradient centrifugation indicated an apparent molecular weight of 390,000 for the solubilized enzyme.

    Conclusions:

    • Gastric (H+ + K+)-ATPase can be effectively solubilized using n-octylglucoside while maintaining its enzymatic function.
    • This study presents the first successful isolation of an active (H+ + K+)-ATPase structure.
    • The findings pave the way for further structural and functional studies of this important ion pump.

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