Stefan Liochev

The role of O2.- in the production of HO.: in vitro and in vivo.

In vitro O2.- reduces Fe(III) to Fe(II), which, in turn, reduces the H2O2, yielding Fe(II)O or HO.. In vivo O2.- increases the supply of free iron by oxidatively attacking the [4Fe-4S] clusters of dehydratases such that they release Fe(II), which can then reduce H2O2. In vivo, O2.- also increases the production of H2O2 by acting as an oxidant toward the dehydratases and toward other cellular reductants.

The oxidation of NADH by tetravalent vanadium

Vanadyl (V(IV)) salts autoxidize in neutral aqueous solution yielding O2- plus vanadate (V(V)) and these, in turn, cause the oxidation of NADH, by a free radical chain reaction. This oxidation of NADH was inhibited by superoxide dismutase, but not by a scavenger of HO.. When H2O2 was present V(IV) caused rapid oxidation of NADH by a process which was unaffected by superoxide dismutase but was inhibited by a scavenger of HO.. This appeared to be dependent upon reduction of H2O2 to OH- plus HO., by V(IV)), followed by oxidation of NADH by HO.. Since there are reductants, within cells, capable of reducing V(V) to V(IV), these reactions are likely to contribute to the toxicity of vanadate.

Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O2- and because vanadate catalyzes the oxidation of NAD(P)H by O2-, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O2-. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.

Superoxide is responsible for the vanadate stimulation of NAD(P)H oxidation by biological membranes

Vandate augments the oxidation of NAD(P)H, but not of NMNH, by rat liver microsomes. Paraquat increases the vanadate effect on NADPH, but not on NADH, oxidation. Substoichiometric levels of NADPH caused the co-oxidation of NADH or NMNH and SOD inhibited in all cases. The ratio of NADH or NMNH co-oxidized per NADPH added allowed estimation of average chain length, which increased as the pH was lowered from 8.0 to 7.1. The initial rate of this co-oxidation of NMNH was a saturating function of the concentration of microsomes, reflecting a decrease in chain length with an increase in number of concomitant reaction chains, and due to increasing radical-radical termination reactions. Mitochondrial outer membranes behaved like the microsomal membranes, but mitochondrial inner membranes catalyzed a rapid oxidation of NADH which could be augmented by vanadate, whose action was enhanced by paraquat and inhibited by antimycin or rotenone. These and related observations support the view that vanadate stimulates NAD(P)H oxidation by biological membranes, not by virtue of interacting with enzymes, but rather by interacting with O-2.

The oxidatin of NADH by vanadate plus sugars

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O2 to O2- and that vanadate plus O2- then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.

Copper increases superoxide dismutase activity in rat liver

Superoxide dismutase (SOD) activity in rat liver cytosol and submitochondrial fractions was characterized as enzymatic and nonenzymatic (due to the SOD-like activity of copper) by four approaches: (i) aerobic NBT2+ (nitroblue tetrazolium) photoreduction in the absence of EDTA; (ii) aerobic NBT2+ photoreduction in the presence of 10(-4)M EDTA; (iii) anaerobic NBT2+ photoreduction; and (iv) o-dianisidine photooxidation. Under normal conditions nonenzymatic SOD activity has been observed only in the intermembrane space. The single subcutaneous injection of rats with CuSO4 solution (5 mg Cu/kg body wt) led to (i) an elevation of the copper level in all submitochondrial fractions; (ii) an increase in enzymatic SOD activity in only cytosol and intermembrane spaces; (iii) the appearance of a new electrophoretic SOD activity band in the intermembrane space preparations; and (iv) the appearance of nonenzymatic SOD-like activity in the outer and inner mitochondrial membranes, and a twofold increase in lipid hydroperoxides. This suggests that the increased nonenzymatic copper in vivo has a prooxidant effect, and does not catalyze the dismutation of O2- as it has been shown in in vitro experiments [E. M. Russanov, S. G. Ljutakova, and S. I. Leutcher (1982) Arch. Biochem. Biophys. 215, 220-229]. The peculiarities of the SOD activity in the intermembrane space are explained by the lysosomal localization of the granular CuZnSOD.

Effects of vanadate on the oxidation of NADH by xanthine oxidase

Vanadate (V(V)) stimulates the oxidation of NADH by xanthine oxidase and superoxide dismutase eliminates the effect of V(V). Paraquat stimulates both the oxidation of NADH by xanthine oxidase and the V(V) enhancement of that oxidation. Xanthine, which is a better substrate for xanthine oxidase than is NADH, causes a V(V)-dependent co-oxidation of NADH which is transient and eliminated by SOD. Urate inhibits the V(V)-stimulated oxidation of NADH by xanthine oxidase or by Rose Bengal plus light. Measurement of rates of both O2- production and V(V)-stimulated NADH oxidation showed that many molecules of NADH were oxidized per O2-. These chain lengths were an inverse function of overall reaction rate. Minimum chain lengths, calculated on the basis of 100% univalent reduction of O2 to O2-, were smaller than measured average chain lengths by a factor of five. All of these results are in accord with the view that V(V) does not directly affect the activity of the enzyme, but rather catalyzes the free radical chain oxidation of NADH by O2-. It was further shown that phosphate was not involved and that the active form of V(V) was orthovanadate, rather than decavanadate.

A study on the mechanism of the vanadate-dependent NADH oxidation.

The mechanism of the vanadate (V(V]-dependent oxidation of NADH was different in phosphate buffers and in phosphate-free media. In phosphate-free media (aqueous medium or HEPES buffer) the vanadyl (V(IV] generated by the direct V(V)-dependent oxidation of NADH formed a complex with V(V). In phosphate buffers V(IV) autoxidized instead of forming a complex with V(V). The generated superoxide radical (O2-) initiated, in turn, a high-rate free radical chain oxidation of NADH. Phosphate did not stimulate the V(V)-dependent NADH oxidation catalyzed by O2--generating systems. Monovanadate proved to be a stronger catalyzer of NADH oxidation as compared to polyvanadate.

Vanadyl-and Vanadate-Induced Lipid Peroxidation in Mitochondria and in Phosphatidylcholine Suspensions

Vanadyl (V(IV] was found to induce rapidly developing lipid peroxidation in intact and sonicated mitochondria as well as in phosphatidylcholine suspension. The ability of vanadate (V(V] to induce lipid peroxidation was much less pronounced compared to that of vanadyl. The peroxidative action of vanadate on phosphatidylcholine much increased in the presence of NADH and ascorbate. Preincubation of vanadate with glucose had the same effect. Vanadyl-induced lipid peroxidation was not essentially influenced by SOD, catalase and ethanol but was completely inhibited by butylated hydroxytoluene. All these effects of vanadyl and vanadate are thought to participate in the insulin-like and other biological actions of vanadium.

Hydroxyl radical is not a significant intermediate in the vanadate-stimulated oxidation of NAD(P)H by O2−

The vanadate-stimulated oxidation of NADH by an enzymatic flux of O2- is inhibited by superoxide dismutase, but not by catalase. Keller et al. (1989, Free Radical Biol. Med. 6, 15-22) observed inhibition by catalase presumably because they used a commercial preparation contaminated with superoxide dismutase. Their proposal, that H2O2 and hydroxyl radical play significant roles in vanadate-stimulation of NAD(P)H oxidation, may be discounted on the basis of these and of previously reported results.