Glutathione Reductase Functions as Vanadate(V) Reductase

"The oxidation of NADPH by vanadate(V) in the presence of glutathione reductase showed typical enzymatic kinetics. The oxidation was inhibited by N-ethylmaleimide, a glutathione reductase inhibitor. Superoxide dismutase had no significant effect on the oxidation, indicating non-involvment of the superoxide radical. The vanadate(V) reduction was found to be a one-electron transfer process. These results suggest a new pathway for vanadate(V) metabolism and a new function of glutathione reductase. Compounds of vanadium(V), such as NaV03 , exert potent toxic effects on a wide variety of biological systems (1-3). Despite intense current activity, however, the biochemical mechanism of vanadate(V) toxicity is still not well understood (1- 14). It is believed that one of the important pathways involves the oxidation (by vanadate(V)) of NAD(P)H (15, 16), but, to our knowledge, there has been no report of the identification of any vanadate(V) reductase. With a view to finding if there exists any vanadate(V) reductase, we have investigated the oxidation of NADPH by vanadate(V) in the presence of glutathione reductase. Glutathione reductase (GSSG-R)2 was selected because our previous studies have shown (17) that GSSG-R can act as a reductase for chromate(VI), which is isoelectronic with vanadate(V). We find that GSSG-R does indeed function as a NADPH-dependent vanadate(V) reductase, thus pointing to a new pathway for the metabolism of vanadate(V), as well as to this property for GSSG-R. MATERIALS AND METHODSSodium metavanadate (NaV03 ), henceforth vanadate, was purchased from Aldrich and its solution was always freshly prepared. The GSSG·R used was from bovine intestinal mucosa and was purchased from Sigma. The oxidation of NADPH was followed photometrically as the decrease in absorbance at 340 nm. The spin trap, 5,5-dimethyl- pyrroline-N-oxide (DMPO), was purchased from Aldrich and used without further purification, since very weak or no spin adduct signal was obtained from the purchased sample when used alone.ESR spectra were obtained at X-band (9.7 GHz) using a Bruker ER200 ESR spectrometer. The magnetic field was calibrated with a self-tracking NMR gaussmeter (Bruker, Model ER035A) and the microwave frequency was measured with a digital frequency counter (Hewlett-Packard, Model 5340A). An ASPECT 2000 computer was used for data storage and analysis. The concentrations given in the figure legends are the final concentrations. All experiments were carried out in the phosphate buffer solution (pH 7.2) and at room temperature."