Noxa1 as a moderate activator of Nox2-based NADPH oxidase

Arch Biochem Biophys. 2012 Mar 1;519(1):1-7. doi: 10.1016/j.abb.2011.12.025. Epub 2012 Jan 8.

Abstract

Noxa1 was discovered as an activating factor for Nox1, an O(2)(-)-generating enzyme. Subsequent studies have shown that Noxa1 is colocalized with Nox2 in several cell types, including vascular cells. Nox2 activation by Noxa1 has been examined in reconstituted model cells. However, little is known about the kinetic properties of Noxa1 in Nox2 activation. In the present study, we used purified cyt.b(558) (Nox2 plus p22(phox)), Rac(Q61L), and Noxo1 to examine the ability of Noxa1 to activate Nox2. In the pure reconstitution system, Noxa1 activated Nox2 with lower efficiency than p67(phox), a canonical activator of Nox2. The EC(50) value of Noxa1 was considerably higher than that of p67(phox). The V(max) value with Noxa1 and Noxo1 was one-third of that with p67(phox) and p47(phox). The EC(50) value of Noxo1 or Rac(Q61L) was also higher when Noxa1 was used. The affinity of FAD for the oxidase and the stability of the active complex were remarkably low when Noxa1 and Noxo1 were used compared with p67(phox) and p47(phox). The stability was not improved by fusion of Noxa1 with Rac(Q61L). These findings show that Noxa1 has quite different kinetic properties from p67(phox) and suggest that Noxa1 may function as a moderate activator of Nox2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport / genetics
  • Adaptor Proteins, Vesicular Transport / metabolism*
  • Adaptor Proteins, Vesicular Transport / pharmacology
  • Cell-Free System
  • Cloning, Molecular
  • Enzyme Activation / drug effects
  • Escherichia coli
  • Flavin-Adenine Dinucleotide / metabolism
  • Humans
  • Immunity, Innate*
  • Kinetics
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • NADPH Oxidase 2
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • Phagocytes / enzymology*
  • Phagocytes / immunology
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Plasmids
  • Protein Binding
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Signal Transduction / physiology*
  • Superoxides / metabolism
  • Transformation, Bacterial

Substances

  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Membrane Glycoproteins
  • NOXA1 protein, human
  • Phosphoproteins
  • Recombinant Fusion Proteins
  • neutrophil cytosol factor 67K
  • Superoxides
  • Flavin-Adenine Dinucleotide
  • CYBB protein, human
  • NADPH Oxidase 2
  • NADPH Oxidases
  • neutrophil cytosolic factor 1
  • Proto-Oncogene Proteins c-akt