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Related Experiment Videos

Poly(adenosine diphosphate ribose) polymerase in Physarum polycephalum.

M D Brightwell, C E Leech, M K O'Farrell

    The Biochemical Journal
    |April 1, 1975
    PubMed
    Summary

    An enzyme in Physarum polycephalum nuclei incorporates NAD+ into poly(ADP-ribose). Enzyme activity fluctuates during the cell cycle, decreasing in early S phase and recovering by late S phase.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Background:

    • The slime mould Physarum polycephalum is a model organism for studying nuclear processes.
    • Poly(ADP-ribose) synthesis is a crucial post-translational modification involved in DNA repair and other cellular functions.

    Purpose of the Study:

    • To characterize an enzyme in Physarum polycephalum nuclei that synthesizes poly(ADP-ribose).
    • To investigate the enzyme's biochemical properties and its activity during the cell cycle.

    Main Methods:

    • Isolation of nuclei from Physarum polycephalum.
    • Enzyme assays measuring the incorporation of [adenine-3H] NAD+ into acid-insoluble products.
    • Determination of optimal pH, temperature, and cofactor requirements.
    • Kinetic analysis (Km, Ki) and effect of inhibitors (nicotinamide).

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  • Assessment of enzyme activity during different phases of the cell cycle.

    • Main Results:

    • The enzyme incorporates NAD+ into poly(ADP-ribose) with optimal activity at pH 8.2 and below 10°C.
    • Magnesium ions (15 mM), dithiothreitol (1 mM), and exogenous DNA stimulate the reaction.
    • Nicotinamide inhibits the enzyme with a Ki of 5.7 μM.
    • Enzyme activity decreases by half in early S phase and returns to pre-mitotic levels by late S phase.
    • The enzyme is stabilized by dithiothreitol and its activity is dependent on DNA.

    Conclusions:

    • A DNA-dependent poly(ADP-ribose) polymerase activity has been identified and characterized in Physarum polycephalum nuclei.
    • The enzyme's activity is cell cycle-regulated, suggesting a role in DNA replication or repair during S phase.