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Tryptic modification of red-cell sodium pump behaviour.

W J Harvey, R Blostein

    Biochimica Et Biophysica Acta
    |April 25, 1986
    PubMed
    Summary

    Tryptic digestion of the human red cell sodium pump affects its hydrolytic activity and alpha-subunit, similar to purified kidney enzymes. This process impacts various ion exchange functions without altering ion stoichiometry.

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    Area of Science:

    • Biochemistry
    • Cellular Biology
    • Membrane Transport

    Background:

    • The sodium pump (Na+/K+-ATPase) is crucial for maintaining cellular ion gradients.
    • Understanding its structure-function relationship is key to cellular homeostasis.
    • In situ studies offer insights into enzyme behavior within its native membrane environment.

    Purpose of the Study:

    • To investigate the effects of controlled tryptic digestion on the human red cell sodium pump in its native membrane.
    • To compare the observed alterations with those previously reported for purified kidney sodium pump enzymes.
    • To elucidate the impact of tryptic digestion on different functional states and transport activities of the sodium pump.

    Main Methods:

    • Utilized inside-out membrane vesicles derived from human red blood cells.
    • Performed controlled tryptic digestion on the sodium pump enzyme in its E1 conformation.
    • Assessed changes in hydrolytic activity, phosphoenzyme levels, and ion transport rates (Na+-K+ exchange, Na+-Na+ exchange, Na+/0 flux).
    • Analyzed subunit cleavage patterns using trypsin and chymotrypsin.

    Main Results:

    • Tryptic digestion caused a greater loss in overall hydrolytic activity than in the phosphoenzyme intermediate.
    • The alpha-subunit was cleaved at the cytoplasmic surface into a ~78 kDa fragment by both trypsin and chymotrypsin.
    • Similar inactivation rates were observed for pump-mediated Na+-K+(Rb+) exchange, (ATP-plus ADP)-dependent Na+-Na+ exchange, and uncoupled Na+ flux (Na+/0 flux).
    • No alteration in Na+:Rb+(K+) stoichiometry was detected after trypsin cleavage.
    • Trypsin similarly affected conformational transitions of both phosphoenzyme and dephosphoenzyme.

    Conclusions:

    • Tryptic digestion of the in situ human red cell sodium pump yields alterations comparable to those of purified kidney enzymes.
    • The cytoplasmic domain of the alpha-subunit is accessible to proteolysis, affecting enzyme activity and ion transport.
    • The sodium pump's conformational dynamics and transport mechanisms are sensitive to proteolytic modification at specific sites.

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