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Related Experiment Videos

Decrease in Na(+)-K(+)-ATPase associated with maturation of sheep reticulocytes.

R Blostein1, E Grafova

  • 1Department of Medicine, McGill University, Montreal, Quebec, Canada.

The American Journal of Physiology
|August 1, 1990
PubMed
Summary
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Sheep red blood cell maturation involves losing sodium pump protein (Na(+)-K(+)-ATPase). This loss occurs partly through energy-dependent endocytosis and shedding of non-functional pump protein into the extracellular environment.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Physiology

Background:

  • The sodium-potassium pump (Na(+)-K(+)-ATPase) is crucial for maintaining cell membrane potential and ion gradients.
  • Red blood cell maturation involves significant changes in protein composition and function, including the regulation of ion transporters.

Purpose of the Study:

  • To investigate the mechanisms underlying the loss of Na(+)-K(+)-ATPase during sheep red blood cell maturation.
  • To characterize the immunological and functional properties of the Na(+)-K(+)-ATPase alpha-subunit during cell maturation and in shed membrane fractions.

Main Methods:

  • Detection of Na(+)-K(+)-ATPase using polyclonal antiserum against purified sheep kidney enzyme.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to identify the alpha-subunit.

Related Experiment Videos

  • Measurement of ouabain binding sites and Na(+)-activated ATP hydrolysis.
  • In vitro culture of immature red blood cells (reticulocytes) to study maturation-associated changes.

    • Main Results:

    • A single major immunologically reactive component corresponding to the alpha-subunit of Na(+)-K(+)-ATPase was detected in both immature and mature sheep red blood cells.
    • Cells separated by density showed a decrease in ouabain binding sites, Na(+)-activated ATP hydrolysis, and detectable alpha-subunit levels.
    • In vitro culture of reticulocytes demonstrated a loss of alpha-subunit associated with reduced ouabain binding, and shed membranous material contained non-functional pump protein.

    Conclusions:

    • Maturation-associated loss of Na(+)-K(+)-ATPase protein in sheep red blood cells involves energy-dependent endocytosis and shedding of pump protein into the extracellular milieu.
    • The shed pump protein is largely devoid of functional activity, indicating a processing step that leads to inactivation.
    • Evidence suggests an intracellular pool of ouabain binding sites in reticulocytes, with transient surface appearance under specific conditions.