Measurement and Characterization of Superoxide Generation from Xanthine Dehydrogenase: A Redox-Regulated
Pathway of Radical Generation in Ischemic Tissues
The enzyme xanthine oxidoreductase
(XOR) is an important source
of oxygen free radicals and related postischemic injury. Xanthine
dehydrogenase (XDH), the major form of XOR in tissues, can be converted
to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by
proteolysis. The conversion of XDH to XO has been assumed to be required
for radical generation and tissue injury. It is also possible that
XDH could generate significant quantities of superoxide, <sup>•</sup>O<sub>2</sub><sup>–</sup>, for cellular signaling or injury;
however, this possibility and its potential ramifications have not
been previously considered. To unambiguously determine if XDH can
be a significant source of <sup>•</sup>O<sub>2</sub><sup>–</sup>, experiments were performed to measure and characterize <sup>•</sup>O<sub>2</sub><sup>–</sup> generation using XDH from chicken
liver that is locked in the dehydrogenase conformation. Electron paramagnetic
resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-<i>N</i>-oxide demonstrated that XDH in the presence of xanthine
produces significant amounts of <sup>•</sup>O<sub>2</sub><sup>–</sup>. NAD<sup>+</sup> and NADH inhibited the generation
of <sup>•</sup>O<sub>2</sub><sup>–</sup> from XDH in
a dose-dependent manner, with NAD<sup>+</sup> exhibiting stronger
inhibition than NADH at low physiological concentrations. Decreased
amounts of NAD<sup>+</sup> and NADH, which occur during and following
tissue ischemia, enhanced the generation of <sup>•</sup>O<sub>2</sub><sup>–</sup> from XDH in the presence of xanthine.
It was observed that XDH-mediated oxygen radical generation markedly
depressed Ca<sup>2+</sup>-ATPase activity of isolated sarcoplasmic
reticulum vesicles from cardiac muscle, and this was modulated by
NAD<sup>+</sup> and NADH. Thus, XDH can be an important redox-regulated
source of <sup>•</sup>O<sub>2</sub><sup>–</sup> generation
in ischemic tissue, and conversion to XO is not required to activate
radical formation and subsequent tissue injury