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An endogenous inhibitory factor for choline acetyltransferase.

C Cozzari, B K Hartman

    Brain Research
    |October 3, 1983
    PubMed
    Summary

    Purifying choline acetyltransferase (CAT) can lead to significant activity loss due to interacting proteins. Low protein concentration and specific factors prevent this inactivation, suggesting a regulatory role in vivo.

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    Area of Science:

    • Biochemistry
    • Neuroscience
    • Enzymology

    Background:

    • Choline acetyltransferase (CAT) is crucial for acetylcholine synthesis.
    • Purification of CAT using carboxymethyl cellulose (CM-cellulose) can lead to substantial enzyme activity loss (up to 95%).
    • This inactivation suggests involvement of endogenous protein interactions.

    Purpose of the Study:

    • To investigate the mechanism of choline acetyltransferase (CAT) inactivation during CM-cellulose chromatography.
    • To identify endogenous factors responsible for CAT activity loss and protection.
    • To explore the physiological relevance of these factors in CAT regulation.

    Main Methods:

    • Partial purification of CAT using CM-cellulose chromatography.
    • Enzyme activity assays under varying protein concentrations and gradient volumes.
    • Mixing experiments with purified inhibitory and activating factors.
    • Temperature dependence studies of the inhibitory factor.

    Main Results:

    • CM-cellulose chromatography separates CAT from endogenous inhibitory and activating factors.
    • An inhibitory factor co-elutes with CAT, causing inactivation unless protein concentration is <0.05 mg/ml.
    • An activating factor protects CAT from the inhibitor's effects.
    • The inhibitor's low temperature dependence suggests it is not a proteolytic enzyme.

    Conclusions:

    • Endogenous protein interactions significantly impact CAT stability during purification.
    • A balance between inhibitory and activating factors may regulate CAT activity in vivo.
    • The identified factors (I50 ≤ 10(-7) M) likely play a physiological role in modulating CAT activity.

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