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Related Experiment Videos

Poly(ADP-ribosyl)ation studies in situ.

D M Duhl, L S Hnilica

    Analytical Biochemistry
    |August 1, 1986
    PubMed
    Summary

    Researchers studied poly(ADP-ribosyl)ation in nuclear proteins using [3H]NAD. They confirmed incorporation into histones and poly(ADP-ribose) polymerase, sensitive to inhibitors.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Cell Biology

    Background:

    • Poly(ADP-ribosyl)ation is a post-translational modification of proteins.
    • Nuclear proteins are targets for poly(ADP-ribosyl)ation.
    • Understanding this process requires methods to study it in situ.

    Purpose of the Study:

    • To investigate the in situ poly(ADP-ribosyl)ation of nuclear proteins.
    • To develop and utilize a method for delivering [3H]NAD to cells for studying incorporation.
    • To determine the kinetics of ADP-ribose incorporation into nuclear proteins.

    Main Methods:

    • Developed a novel method for intracellular delivery of [3H]NAD.
    • Quantified the kinetics of [3H]NAD uptake and incorporation into nuclear proteins.
    • Utilized polyacrylamide gel electrophoresis and immunoreaction with anti-(ADP-ribose) antibodies for protein identification.

    Main Results:

    • Demonstrated successful delivery of [3H]NAD to cells in vitro.
    • Observed significant incorporation of ADP-ribose into the histone fraction.
    • Confirmed radioactivity association with poly(ADP-ribose) polymerase and histones, sensitive to 3-aminobenzamide.

    Conclusions:

    • The study successfully established a method to study in situ poly(ADP-ribosyl)ation.
    • Confirmed that histones and poly(ADP-ribose) polymerase are major acceptors of poly(ADP-ribose) in the nucleus.
    • Validated the findings through biochemical and immunological techniques.

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