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Related Experiment Videos

Solubilization of human prostatic 5 alpha-reductase.

B Houston, G D Chisholm, F K Habib

    Journal of Steroid Biochemistry
    |April 1, 1985
    PubMed
    Summary

    A new assay detects 5 alpha-reductase activity, enabling the development of a method to solubilize this enzyme from human prostate tissue. Lubrol PX effectively solubilized 70% of active 5 alpha-reductase.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Molecular Biology

    Background:

    • 5 alpha-reductase is crucial for androgen metabolism.
    • Solubilization of membrane-bound enzymes like 5 alpha-reductase is challenging.
    • Understanding enzyme activity requires stable and active preparations.

    Purpose of the Study:

    • To develop a sensitive assay for 5 alpha-reductase.
    • To establish a method for solubilizing human prostatic 5 alpha-reductase.
    • To identify optimal conditions for enzyme stabilization and solubilization.

    Main Methods:

    • Developed a sensitive assay detecting ≥0.2 U/sample of 5 alpha-reductase activity.
    • Optimized homogenization to release 95% of enzyme into microsomal fraction.
    • Investigated detergent effects on enzyme activity and solubilization, focusing on Lubrol PX.

    Main Results:

    • A stabilization buffer (0.1 M sodium citrate, 0.1 M KCl, 20% glycerol, 0.5 mM NADPH, 1 μM testosterone) was identified.
    • Certain detergents (Triton X-100, Lubrol PX, Nonidet P-40) inhibited enzyme activity in a dose-dependent manner.
    • Lubrol PX at <1.1 mg/ml yielded significant active solubilized enzyme; 3 extractions with 0.8 mg/ml Lubrol PX solubilized 70% of active enzyme.

    Conclusions:

    • A sensitive and reproducible assay for 5 alpha-reductase was established.
    • Optimal conditions for stabilizing and solubilizing human prostatic 5 alpha-reductase were determined.
    • Lubrol PX is an effective detergent for recovering active 5 alpha-reductase.

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