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Related Experiment Videos

Human saturated steroid 6alpha-hydroxylase

R Dombroski1, M L Casey, P C Macdonald

  • 1Department of Obstetrics-Gynecology, University of Texas Southwestern Medical School, Dallas 75235, USA.

The Journal of Clinical Endocrinology and Metabolism
|May 1, 1997
PubMed
Summary
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This study identifies a distinct saturated steroid 6alpha-hydroxylase in human tissues, crucial for metabolizing anxiolytic steroids like 5alpha-dihydroprogesterone (5alpha-DHP). This enzyme is not a cytochrome P450, differentiating it from other steroid hydroxylases.

Area of Science:

  • Biochemistry
  • Steroid Metabolism
  • Enzymology

Background:

  • Extrahepatic human tissues metabolize progesterone and 5alpha-dihydroprogesterone (5alpha-DHP) into anxiolytic/anesthetic steroids.
  • A distinct saturated steroid 6alpha-hydroxylase is responsible for further metabolism of these steroids.
  • This enzyme is separate from cytochrome P450 6alpha-hydroxylases involved in other steroid transformations.

Purpose of the Study:

  • To further characterize the saturated steroid 6alpha-hydroxylase in extrahepatic human tissues.
  • To investigate the enzyme's activity, substrate specificity, and localization using T47-D human breast cancer cells.

Main Methods:

  • Utilized microsome-enriched preparations from T47-D human breast cancer cells as the enzyme source.
  • Assayed enzyme activity with varying incubation times, protein concentrations, and pH.

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  • Determined kinetic parameters (Km, Vmax) and tested substrate efficiency using specific steroid precursors.
  • Investigated the effect of cytochrome P450 inhibitors on enzyme activity.

    • Main Results:

    • Saturated steroid 6alpha-hydroxylase activity was highest in microsome-enriched fractions.
    • Enzyme activity was linear with time (up to 30 min) and protein concentration (0.05-0.5 mg/mL).
    • Optimal pH range was broad (6.0-8.0), with both NADH and NADPH supporting activity.
    • 5alpha-pregnan-3beta-ol-20-one and 5alpha-pregnan-3alpha-ol-20-one were efficient substrates with indistinguishable kinetic parameters (Km ≈ 3.5 μM, Vmax ≈ 150 pmol/min/mg).
    • 5alpha-androstane-3beta,17beta-diol and 5alpha-androstane-3alpha,17beta-diol were less efficient substrates.
    • Cytochrome P450 inhibitors did not significantly affect 6alpha-hydroxylation, confirming the enzyme is not a P450.

    Conclusions:

    • Human saturated steroid 6alpha-hydroxylase is a distinct enzyme, not a cytochrome P450.
    • It efficiently metabolizes specific 5alpha-pregnan-3-ol-20-one isomers, which are precursors to major urinary metabolites.
    • T47-D cells provide a viable source for studying this enzyme's properties.