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Related Experiment Videos

Pituitary progesterone 5 alpha-reductase: solubilization and partial characterization.

P J Bertics, H J Karavolas

    Journal of Steroid Biochemistry
    |June 1, 1985
    PubMed
    Summary

    Researchers solubilized progesterone 5 alpha-reductase from rat pituitary glands using n-octyl glucoside (OG)-KCl extraction. This method yielded greater enzyme activity and preserved kinetic properties, indicating successful stabilization of the enzyme.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Endocrinology

    Background:

    • The anterior pituitary gland plays a crucial role in hormonal regulation.
    • Microsomal enzymes, such as progesterone 5 alpha-reductase, are vital for steroid metabolism.
    • Understanding enzyme properties is key to elucidating cellular functions.

    Purpose of the Study:

    • To solubilize and characterize progesterone 5 alpha-reductase from female rat anterior pituitary microsomes.
    • To investigate the kinetic and physical properties of the solubilized enzyme.
    • To compare the properties of the solubilized enzyme with the native microsomal enzyme.

    Main Methods:

    • Enzyme solubilization using n-octyl glucoside (OG)-KCl or digitonin-KCl extraction.
    • Kinetic analysis to determine apparent Km for progesterone.

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  • Enzyme stability assays including dialysis and assessment of phospholipid effects.
  • Inhibition studies using metal chelators and sulfhydryl blocking agents.
  • Evaluation of flavin involvement in the enzymatic reaction.

    • Main Results:

    • OG-KCl extraction yielded higher specific activity compared to digitonin-KCl.
    • Solubilized enzyme exhibited similar Km for progesterone as the microsomal form, indicating preserved kinetics.
    • The enzyme demonstrated stability after dialysis and could be stimulated by phospholipids.
    • Progesterone 5 alpha-reductase was unaffected by metal chelators but inhibited by p-chloromercuribenzoic acid.
    • High flavin levels and analogs inhibited the enzyme activity.

    Conclusions:

    • The OG-KCl method is effective for solubilizing active and stable progesterone 5 alpha-reductase from rat anterior pituitary.
    • The solubilization process maintains the enzyme's kinetic characteristics.
    • The enzyme's properties suggest it is a sulfhydryl-dependent enzyme, potentially not involving flavins as hydride carriers.