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Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8.

H L Santos1, R P Lamas, P Ciancaglini

  • 1Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.

Brazilian Journal of Medical and Biological Research = Revista Brasileira De Pesquisas Medicas E Biologicas
|March 12, 2002
PubMed
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This study introduces a faster method for purifying Na,K-ATPase (sodium-potassium adenosine triphosphatase) using only the C12E8 detergent. The new technique significantly reduces purification time while maintaining enzyme activity and stability.

Area of Science:

  • Biochemistry
  • Enzymology
  • Membrane Proteins

Background:

  • Traditional purification of Na,K-ATPase often involves multiple detergents like SDS, CHAPS, or CHAPSO.
  • These methods can be time-consuming and may affect enzyme activity.
  • Efficient purification is crucial for studying enzyme kinetics and structure.

Purpose of the Study:

  • To develop a streamlined purification protocol for Na,K-ATPase.
  • To evaluate the efficacy of using only C12E8 detergent for solubilization and purification.
  • To characterize the activity and properties of the purified enzyme.

Main Methods:

  • Solubilization of Na,K-ATPase-rich membrane fragments from rabbit kidney outer medulla using C12E8.
  • Purification via a single-step gel filtration chromatography on Sepharose 6B.

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  • Analysis using non-denaturing PAGE and enzyme activity assays.

    • Main Results:

    • Achieved 98% activity recovery with C12E8 under optimized conditions (4°C, 1 mg/ml C12E8).
    • Purified enzyme showed a single protein band on PAGE with phosphomonohydrolase activity.
    • Estimated molecular mass of 320 kDa, optimal pH of 7.5, and exhibited negative cooperativity for ATP binding.
    • Demonstrated significant inhibition by ouabain, oligomycin, and sodium vanadate.

    Conclusions:

    • Na,K-ATPase can be effectively solubilized and purified using only C12E8 detergent.
    • This method offers a significant reduction in purification time and complexity.
    • The purified enzyme retains its activity and characteristic properties, consistent with an (alphabeta)2 quaternary structure.