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Hypoxanthine phosphoribosyltransferase from human brain: purification and partial characterization.

G W Smithers, W J O'Sullivan

    Biochemical Medicine
    |August 1, 1984
    PubMed
    Summary

    Researchers purified hypoxanthine phosphoribosyltransferase (HPRT) from human brain using heat denaturation and GMP-Sepharose chromatography. The enzyme is a tetramer with three charge variants, suggesting complex regulation of HPRT activity.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Neuroscience

    Background:

    • Hypoxanthine phosphoribosyltransferase (HPRT) is a crucial enzyme in purine metabolism.
    • Understanding HPRT's structure and variants is vital for comprehending its role in cellular function and disease.

    Purpose of the Study:

    • To develop a facile and rapid purification method for human brain hypoxanthine phosphoribosyltransferase.
    • To characterize the molecular properties, including subunit composition and charge heterogeneity, of the purified enzyme.

    Main Methods:

    • Heat denaturation of extraneous proteins.
    • GMP-Sepharose affinity chromatography for enzyme purification.
    • Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for subunit molecular weight estimation.

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  • Gradient polyacrylamide gel electrophoresis for native molecular weight determination.
  • Nondenaturing PAGE, electrofocusing, and chromatofocusing for charge variant analysis.

    • Main Results:

    • A homogeneous preparation of human brain hypoxanthine phosphoribosyltransferase was obtained.
    • The subunit molecular weight was determined to be 24,000 Da by SDS-PAGE.
    • The native molecular weight was estimated at approximately 100,000 Da, indicating a tetrameric structure.
    • Three major charge variant forms with acidic isoelectric points (pI) of approximately 5.7, 5.5, and 5.0 were identified.

    Conclusions:

    • The purification strategy effectively isolates homogeneous human brain hypoxanthine phosphoribosyltransferase.
    • Human brain hypoxanthine phosphoribosyltransferase exists as a tetramer, similar to the erythrocyte enzyme.
    • The presence of multiple charge variants suggests post-translational modifications or genetic polymorphisms influencing enzyme function.