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A microassay for UDP-glucose dehydrogenase.

R B Burrows, C Cintron

    Analytical Biochemistry
    |April 15, 1983
    PubMed
    Summary

    A new fluorometric assay for UDP-glucuronic acid enables sensitive determination of UDP-glucose dehydrogenase activity. This method allows enzyme quantification in small tissue samples and reveals no distinct enzyme forms in rabbit tissues.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Analytical Chemistry

    Background:

    • UDP-glucose dehydrogenase (UGDH) is a key enzyme in glucuronidation pathways.
    • Existing spectrophotometric assays for UGDH lack sensitivity for small sample sizes.
    • A sensitive assay for UDP-glucuronic acid is crucial for UGDH activity determination.

    Purpose of the Study:

    • To adapt a UDP-glucuronic acid assay for sensitive UGDH activity measurement.
    • To determine kinetic properties of UGDH from rabbit tissues.
    • To investigate the presence of different UGDH forms in various rabbit tissues.

    Main Methods:

    • Utilized a fluorometric assay for UDP-glucuronic acid, a product of UGDH.
    • The assay involves forming D-glucuronosyl benzo(a)pyrene from UDP-glucuronic acid and 3-hydroxybenzo(a)pyrene.
    • Enzyme activity was measured after removing unreacted substrate via chloroform-methanol extraction.

    Main Results:

    • The fluorometric assay demonstrated approximately 500-fold higher sensitivity compared to spectrophotometric methods.
    • The assay allows UGDH quantification in milligram quantities of connective tissue.
    • Kinetic properties of UGDH from rabbit tissue were determined.
    • No evidence of distinct UGDH forms was found in rabbit liver, cartilage, or corneal stroma.

    Conclusions:

    • The developed fluorometric assay provides a highly sensitive method for UGDH activity determination.
    • This assay is suitable for analyzing enzyme activity in limited biological samples.
    • UGDH appears to be a single form across different rabbit tissue types examined.

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