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Tumor glutaminase purification

J A Segura1, J C Aledo, S Gómez-Biedma

  • 1Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Málaga, Spain.

Protein Expression and Purification
|June 1, 1995
PubMed
Summary

Researchers developed two novel methods to purify glutaminase from Ehrlich tumor cells, achieving significantly higher purity and enzyme stability. These techniques enhance the isolation of this crucial enzyme for further study.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Cell Biology

Background:

  • Glutaminase is an important enzyme involved in cellular metabolism.
  • Efficient purification of glutaminase from Ehrlich tumor cells is crucial for biochemical and functional studies.
  • Previous purification methods for Ehrlich tumor cell glutaminase were suboptimal, limiting research.

Purpose of the Study:

  • To develop and optimize two distinct, highly efficient purification schemes for glutaminase from Ehrlich tumor cells.
  • To achieve a higher degree of purity than previously reported.
  • To characterize the purified glutaminase and validate its identity.

Main Methods:

  • Purification Scheme 1: Utilized conventional and high-performance liquid chromatography (HPLC), including ion-exchange, hydroxyapatite, and hydrophobic interaction chromatography, following mitochondrial isolation and solubilization.

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  • Purification Scheme 2: Employed native polyacrylamide gel electrophoresis (PAGE), in situ activity staining, and electroelution for rapid isolation.
  • Characterization: Determined apparent molecular weight using gel filtration and sucrose density gradient ultracentrifugation; validated enzyme identity using immunoblot analysis with specific antibodies.

    • Main Results:

    • Scheme 1 yielded a 37-fold increase in glutaminase purification compared to previous reports.
    • Scheme 2 provided a simple, rapid isolation with a 10-fold higher purification, minimizing proteolytic inactivation.
    • Immunoblot analyses confirmed the identity of the purified enzyme, showing cross-reactivity with antibodies against kidney glutaminase.

    Conclusions:

    • Two effective and distinct purification strategies for Ehrlich tumor cell glutaminase have been established.
    • These methods significantly improve enzyme purity and stability, facilitating further research.
    • The purified enzyme's identity was confirmed, providing a reliable source for biochemical and immunological investigations.