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Related Experiment Videos

Iron-dependent uptake of ascorbate into isolated microsomes.

B Peterkofsky, G Tschank, C Luedke

    Archives of Biochemistry and Biophysics
    |April 1, 1987
    PubMed
    Summary

    Microsomes concentrate vitamin C (ascorbate) via an iron-dependent process, crucial for prolyl hydroxylase activity in collagen synthesis. This uptake mechanism protects ascorbate from oxidation.

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    Area of Science:

    • Biochemistry
    • Cell Biology
    • Molecular Biology

    Background:

    • Chick embryo limb bone microsomes previously suggested to concentrate ascorbate.
    • Prolyl hydroxylase activity is essential for collagen synthesis.
    • Ascorbate (vitamin C) is a critical cofactor for prolyl hydroxylase.

    Purpose of the Study:

    • To investigate the mechanism of ascorbate uptake into microsomes.
    • To determine the role of iron in ascorbate transport and its functional significance.
    • To compare ascorbate kinetics in endogenous and exogenous prolyl hydroxylase systems.

    Main Methods:

    • Measurement of [14C]ascorbate uptake into chick embryo limb bone and L-929 cell microsomes.
    • Kinetic analysis of ascorbate hydroxylation using endogenous and solubilized prolyl hydroxylase.
    • Competition assays with unlabeled ascorbate and dehydroascorbate.
    • Functional assays involving iron preincubation and ascorbic acid oxidase treatment.

    Main Results:

    • Apparent Km for ascorbate in endogenous hydroxylation was significantly lower than with solubilized enzyme, suggesting concentrative uptake.
    • [14C]Ascorbate uptake into microsomes was iron-dependent (Fe2+ or Fe3+), time, temperature, and microsome concentration-dependent, and saturable.
    • Ascorbate uptake occurred without prior oxidation, as dehydroascorbate did not compete.
    • Iron was required for functional ascorbate translocation into microsomes, protecting it from external oxidation.

    Conclusions:

    • Microsomal ascorbate uptake is an active, iron-dependent process.
    • This iron-dependent transport mechanism is essential for maintaining intracellular ascorbate levels for prolyl hydroxylase activity.
    • The findings elucidate a novel aspect of vitamin C metabolism and its role in collagen biosynthesis.

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