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Related Experiment Videos

Hepoxilin A3 synthase.

Santosh Nigam1, Maria-Patapia Zafiriou

  • 1Eicosanoid and Lipid Research Division, Centre of Experimental Gynecology and Breast Research, University Medical Centre Berlin, Charité-Campus Benjamin Franklin, D-12200 Berlin, Germany. nigam@zedat.fu-berlin.de

Biochemical and Biophysical Research Communications
|October 4, 2005
PubMed
Summary

A novel leukocyte-type 12S-lipoxygenase (12S-LOX) enzyme from rat insulinoma cells possesses intrinsic hepoxilin A3 (HXA3) synthase activity, clarifying HXA3 biosynthesis. Glutathione peroxidase activity modulates HXA3 formation by competing for the same substrate.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Hepoxilins are epoxy-hydroxy fatty acids derived from 12S-hydroperoxyeicosatetraenoic acid (12S-HpETE).
  • Hepoxilin A3 (HXA3) has diverse biological activities, but its synthesis pathway remains unclear.
  • 12S-lipoxygenase (12S-LOX) enzymes are implicated in eicosanoid metabolism.

Purpose of the Study:

  • To investigate the biosynthetic mechanism of hepoxilin A3 (HXA3).
  • To identify and characterize the enzyme responsible for HXA3 synthesis.
  • To explore the regulation of HXA3 formation.

Main Methods:

  • Isolation, cloning, and characterization of rat 12S-lipoxygenase (12S-LOX) from RINm5F cells.
  • Coimmunoprecipitation assays to link HXA3 synthase activity with 12S-LOX.

Related Experiment Videos

  • Site-directed mutagenesis of rat 12S-LOX to assess enzyme activity and substrate specificity.
  • Enzyme assays to quantify HXA3 synthase activity.

    • Main Results:

    • A leukocyte-type 12S-LOX was identified in rat insulinoma cells with intrinsic HXA3 synthase activity.
    • HXA3 synthase activity was confirmed to be associated with 12S-LOX via coimmunoprecipitation and recombinant enzyme assays.
    • Mutagenesis studies revealed that 12-lipoxygenation activity is crucial for HXA3 synthesis, while 15-lipoxygenation impairs it.
    • Cellular glutathione peroxidases (cGPx and PHGPx) compete with HXA3 synthase for the 12S-HpETE substrate.

    Conclusions:

    • Leukocyte-type 12S-lipoxygenase (12S-LOX) is identified as the enzyme responsible for hepoxilin A3 (HXA3) biosynthesis.
    • The positional specificity of 12S-LOX is critical for its HXA3 synthase activity.
    • Cellular peroxide tone, regulated by glutathione peroxidases, influences HXA3 production by modulating substrate availability.