Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Nucleosidediphosphate kinase from Ehrlich ascites tumor cells.

K Koyama, M Yokoyama, T Koike

    Journal of Biochemistry
    |April 1, 1984
    PubMed
    Summary

    Researchers purified a novel nucleoside diphosphate kinase (NDP-kinase) from Ehrlich ascites tumor cells. This enzyme forms a high-energy phosphate intermediate, crucial for its catalytic function in cellular energy transfer.

    Related Concept Videos

    You might also read

    Related Articles

    Articles linked to this work by shared authors, journal, and citation graph.

    Sort by
    Same author

    Cryptic pericentric inversion of chromosome 17 detected by fluorescence in situ hybridization study in familial Miller-Dieker syndrome.

    American journal of medical genetics·1997
    Same author

    beta-cell function in normal rats made chronically hyperleptinemic by adenovirus-leptin gene therapy.

    Diabetes·1997
    Same author

    [Laparoscopic retrieval of a dislocated ventriculoperitoneal shunt catheter: report of three cases and a review of the literature].

    No shinkei geka. Neurological surgery·1997
    Same author

    Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation.

    Proceedings of the National Academy of Sciences of the United States of America·1997
    Same author

    Association between Ala54Thr substitution of the fatty acid-binding protein 2 gene with insulin resistance and intra-abdominal fat thickness in Japanese men.

    Diabetologia·1997
    Same author

    Antinociceptive components of Ganoderma lucidum.

    Planta medica·1997

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzymology

    Background:

    • Ehrlich ascites tumor cells (EAT cells) are a model system for studying cellular metabolism and protein function.
    • Nucleoside diphosphate kinases (NDP-kinases) play a vital role in cellular energy metabolism by catalyzing the transfer of a terminal phosphate group.
    • Understanding the properties of NDP-kinases from tumor cells can provide insights into cancer biology and potential therapeutic targets.

    Purpose of the Study:

    • To highly purify and characterize a phosphate-incorporating protein from EAT cell cytosol.
    • To identify the purified protein and elucidate its enzymatic activity and properties.
    • To investigate the mechanism of action, specifically the formation and role of a phosphoenzyme intermediate.

    Main Methods:

    Related Experiment Videos

  • Purification of the target protein using biochemical techniques, with [32P]phosphorylation as a marker.
  • Nitrocellulose membrane assay for quantitating the [32P]phosphorylated protein during purification.
  • Enzyme activity assays to confirm NDP-kinase function.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination.
  • Amino acid analysis to determine the protein's composition.
  • Kinetic studies to characterize the formation and utilization of the phosphoenzyme intermediate.

    • Main Results:

    • A phosphate-incorporating protein was successfully purified from EAT cell cytosol.
    • The purified protein exhibited NDP-kinase activity and shared characteristics with mammalian NDP-kinases.
    • The enzyme has an estimated molecular weight of 76,000 daltons, composed of two subunits (18,000 and 20,000 daltons).
    • Amino acid analysis revealed high percentages of glycine (9.8%) and lysine (9.0%).
    • A [32P]phosphoenzyme intermediate was rapidly formed with [gamma-32P]ATP and Mg2+ at pH 7.5 and low temperatures.
    • This phosphoenzyme intermediate was shown to be a high-energy phosphate compound, capable of transferring its phosphate to ADP to form ATP, confirming its role in NDP-kinase catalysis.

    Conclusions:

    • The purified protein is a mammalian NDP-kinase from EAT cells.
    • The enzyme functions via a mechanism involving a high-energy phosphoenzyme intermediate.
    • This characterization provides a molecular basis for understanding NDP-kinase function in tumor cells and energy metabolism.