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

Free Radical Biology & Medicine, Vol.5, pp. 349-354, 1988

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Original Contribution A STUDY

ON THE

MECHANISM OF THE VANADATE-DEPENDENT NADH OXIDATION

STEFAN I. LIOCHEV* and EKATERINA A. IVANCHEVA Department of Bieenergetics, Instituteof Physiology,BulgarianAcademyof Sciences, Acad. G. BonchevSt., bl. 23, 1113 Sofia, Bulgaria (Received 20 January 1988; Revised 31 March 1988; Accepted 13 April 1988)

AbstractmThe mechanism of the vanadate (V~v))-dependentoxidation of NADH was different in phosphate buffers and in phosphate-free media. In phosphate-free media (aqueous medium or HEPES buffer) the vanadyl (Vav)) generated by the direct V~v)-dependentoxidation of NADH formed a complex with V~v). In phosphate buffers V,w) autoxidized instead of forming a complex with V~v,. The generated superoxide radical (02-) 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. Keywords--Vanadate, Vanadyl, Superoxide radical, NADH, SOD, Vanadate-dependent NADH oxidation, Free radical

INTRODUCTION

are observations that V~v)-dependent NAD(P)H oxidation catalyzed by biomembranes also occurs in phosphate-free buffers. 1'9 Dart and Fridovich m4 have reported that the V¢v)-dependent NAD(P)H oxidation catalyzed by xanthine + xanthine oxidase does not require phosphate, and that monovanadateiS-more active than polyvanadate. The present study was undertaken to cast further light on these problems.

It is known that vanadate (V(v)) catalyzes the oxidation of NAD(P)H by biomembranes,l-l° xanthine oxidase,ll xanthine + xanthine oxidase, 12-~4photosensitizers + light, ~2KO: 12or reducing sugars. 15There are data ~°,~2.16 that V~v)-dependent NAD(P)H oxidation is a 02-initiated free radical chain process. Vijaya and Ramasarma )7 have reported a V~v)-dependent NADH oxidation in the absence of biomembranes or other exogenous systems. According to these authors, it is a matter of a direct NADH oxidation by V~v), which follows second-order kinetics. This, however, seems unreasonable because the SOD sensitivity of the process suggests O:--dependent chain reaction. Furthermore the rate constant calculated by Vijaya and Ramasarma ~7 is too low to explain the relatively high rates of NADH oxidation. Opinions also differ concerning the active form of vanadate in the vanadateinduced NAD(P)H oxidation--monovanadate or polyvanadate (decavanadate) as well as the possible necessity of phosphate for this process. Some authors claim that only decavanadate catalyzes NAD(P)H oxidation in phosphate buffers, that is, after Vtv>-phosphate complex formation.5-8'17 On the other hand, there

MATERIALS AND METHODS

NADH oxidation and V~v) reduction were recorded by a Specord UV-VIS spectrophotometer at 340 nm and 750 nm, respectively. Oxygen consumption was measured polarographically by a Clark electrode. Unless otherwise stated, freshly prepared colorless 20 mM stock solutions of vanadate were used. The reagents used were: Ammonium metavanadate (Sigma), vanadyl sulphate (Aldrich), NADH and catalase (Boehringer), KH2PO4 and KOH (Merch), HEPES and Rose Bengal (Serva), mannitol (BDH). Cu,Zn-Superoxide dismutae (SOD) was generously provided by Professor I. Fridovich, Department of Biochemistry, Duke University Medical Center, Durham, NC, USA.

*Author to whom correspondenceshouldbe addressed. 349

350

S . I . LtOCXEV and E. A. IVANCHEVA

rate of this process and the free radical chain length decrease with the concentration of V~v)and NADH and with increasing pH. SOD strongly inhibited the V(v~-dependent NADH oxidation (Fig. la, line 4; Fig. Ib, line 4) and changed the kinetics of the NADH oxidation, making it similar to that of a normal chemical reaction. Catalase (5 ag/ ml), mannitol (50 mM) and ethanol (1%) (data not shown) suppressed this process but to a lower extent. Boiled SOD and catalase were essentially ineffective. The SOD inhibition of the V~v)-dependent NADH oxidation is consistent with the results of Vijaya and Ramasarma t7 but these authors failed to find an effect of catalase and "OH scavengers. The V(v)-dependent oxidation of NADH was stimulated by phosphate (Fig. 2). In 100 mM HEPES buffer, pH 7,0 or in water adjusted to pH 7.0 oxidation of NADH was not observed in the course of 10 min (Fig. 2, line 1). In the same media but with pH 6.0 oxidation of NADH occurred at a very low rate after a long lag (Fig. 2, line 6). In media containing increasing concentrations of phosphate the lag decreased

RESULTS

The V(v)-dependent oxidation of NADH in the presence of 100 mM phosphate buffer is shown in Figure 1. The kinetics are typical neither of first- nor of second-order reactions. NADH oxidation exhibited sigmoidal kinetics, that is, lag period, acceleration, approach to maximum, and deceleration. Such kinetics are characteristic of chain reactions, where the lag is probably determined by the time necessary for accumulation of the active species, and the deceleration is due to the depletion of substrate. With the decrease of V(v) concentration and with the increase of pH of the buffer (Figs. I a, b), the lag increased and the maximum rate of the reaction decreased. The lag also increased and the reaction rate decreased with decreasing NADH concentration (data not shown). That the rate of the V(v)-dependent oxidation of NADH was affected by the concentrations of V~v) and NADH as well as by pH was not surprising. Our earlier data '° showed that the biomembrane-catalyzed V(v)-dependent oxidation of NAD(P)H has a free radical nature and that both the

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Fig. 1. Oxidation of NADH by V~v). Reaction mixtures contained 0.2 mM NADH and 100 mM potassium phosphate buffer at pH 6.0 (a) and pH 7.0 (b). Reactions were started with the addition of vanadate: (a)~0.025 mM (line 1); 0.250 mM (line 2); 0.500 mM (line 3); 0.500 mM after l #g/ml SOD (line 4); (b) - 0 . 2 5 0 mM (line I); 0.500 mM (line 2); 0.750 mM (line 3); 0.750 mM after 1 a g / m l SOD (line 4).

Vevj-dependent NADH oxidation

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Time (rain) Fig. 2. Effect of phosphate on the V~v,-dependent NADH oxidation. Reactions were started with the addition of 0.750 mM V~vj to 0.2 mM NADH in: 100 mM HEPES buffer, pH 7.0 or water adjusted to pH 7.0 (line 1); 5 raM, 25 raM, 40 mM and 100 mM potassium phosphate buffer, pH 7.0 (lines 2, 3, 4, 5); 100 mM HEPES buffer or water, pH 6.0 (line 6).

and the reaction rate increased (Fig. 2, lines 2, 3, 4, 5). These results are in accordance with the data of Vijaya and Ramasarma '7 that phosphate stimulates the nonenzymic V¢v)-dependent NADH oxidation. We have found that V(,v, catalyzes a rapid NADH oxidation and that this SOD-sensitive chain process occurs after a lag determined by the V,v~ accumulation.'6 By analogy, we assumed that the lag in the V~v~dependent NADH oxidation was determined by the Vav) accumulation. In fact, when V¢~v~was added to the NADH- and Vcv)-containing phosphate buffers, NADH oxidation occurred so rapidly that only the end of the reaction could be recorded (Fig. 3, line 1). Addition of Vewl to NADH- and V(v~-containing HEPES buffer initiated an oxidation of NADH, which also

occurred without a lag but its rate was at least 30 times lower than that in phosphate buffer (Fig. 3, line 2). The same was observed in water (data not shown). In both cases NADH oxidation was almost completely inhibited by SOD (data not shown). The kinetics of the V(v)-dependent NADH oxidation, the inhibitory effect of SOD and the synergism between V(v) and Vow) in stimulating the NADH oxidation suggest a direct oxidation of some NADH by~V(v) as the O2- generated by autoxidation of the reduced vanadate initiates, in turn, a free radical chain oxidation of NADH. Bearing in mind the contradictory data about the necessity of phosphate for the V(v)-dependent NADH

Table

Vv Vlv

1. Effects

of

V~v) on Rose Bengal-Photosensitized Oxidation of NADH RB + light

V.V V,IV Medium A HEPES buffer, 10mM HEPES buffer, 10mM + 2 ~tg/ml SOD HEPES buffer, 100mM Phosphate buffer, 10mM Phosphate buffer, 10mM + 2/~g/ml S O D

RB + light + Vrv) AAm

0.007

0.340

0.007 0.007 0.008

0.010 0.305 0.345

0.008

0.010

0.019 0.018 0.019

0.290 0.295 0.305

B

lrnin o

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Fig. 3. Acceleration of the V(v,-dependent NADH oxidation by Vav~. Reaction mixtures contained 0.2 mM NADH in 100 mM potassium phosphate buffer, pH 6.0 (line 1) or 100 mM HEPES buffer, pH

6.0 (line 2). Additions: 0.3 mM V~v,; 0.15 mM V~lvj.

HEPES buffer, 10raM Phosphate buffer, 10raM Phosphate buffer, 100raM

Aliquots (2 nil) containing 2 #M Rose Bengal (RB) and 0.2 mM NADH with or without V~v~ in phosphate or HEPES buffer, pH 6.0 were placed in cuvettes (1 cm light path) and irradiated with light by a 20 W fluorescent mhe. The concentration of Vw) was 0.4 mM (A) and 0.1 mM (B). The light intensity in (B) was increased in such a way as to make the rate of the V~v)stimulated photosensitir~-,,d NADH oxidation approximately equal in both (A) and (B). AA~0 was obtained by subtracting A3~ of the irradiated samples from A~0 of the dark controls.

352

S . I . L1OCHEV and E. A. IVANCHEVA

oxidation, 5,~4.~7 we supposed that phosphate was needed for the vanadyl-dependent 05- generation and not for the Os--initiated V~v)-dependent NADH oxidation. This assumption was confirmed by the results summarized on Table 1. It is seen that the effect of V~v) on the NADH oxidation, photosensitized by Rose Bengal + light, an exogenous V~v~-independentsource of 05-15, was almost the same with both buffers used as well as in water (data not shown). SOD (2 #g/ml) completely abolished the stimulant effect of V~v) on NADH oxidation on all media. Previously, we observed that incubation of VIv~with sugars or mixing V~iv) and V~v) in water caused the formation of a blue-green compound which was probably a V~v~ - V(iv) complex. Is In the present experiments we found that a bluegreen compound, absorbing at 750 nm, was formed both on mixing V
0.6

0.4

peared when V~v) was incubated with NADH in 100 mM HEPES (Fig. 5). On incubation of V,v> with NADH in 100 mM phosphate buffer neither an increase of the 750 nm absorption nor the appearance of bluegreen color was observed (Fig. 5). It is concluded that in the case of Vcrv)formation in V(v~-containing medium in the presence of oxygen, vanadyl either forms a stable complex with V(v~ or autoxidizes. The 02- generated in the presence of phosphate could, in turn, initiate a Vw)-dependent NADH oxidation via a free radical chain mechanism. Phosphate stimulates the V(v>-dependent NADH oxidation, inhibiting the formation of a stable V(v) - V(tv) complex and does not preclude the V(w) autoxidation. In agreement with such an interpretation V(w) virtually autoxidized in 100 mM phosphate buffer, pH 7.0, as judged by the O5 consumption (data not shown). Acidification of a concentrated solution of colorless monovanadate leads to a rapid polymerization of vanadate to decavanadate accompanied by the appearance of yellow color. ~s A 20 mM freshly prepared colorless metavanadate solution was acidified with diluted HC1 to pH 4.0, which resulted in the appearance of deep yellow color and 5 min later neutralized to pH 7.0 with diluted NaOH. The UV spectra of the yellow solution, containing decavanadate and of the colorless monovanadate solution are shown in Fig. 6a. The polymerization of vanadate caused a decrease of the absorption in the far UV region and an increase of the absorption in the near UV region. Similar changes in the interconversion of decavanadate to monovanadate have been reported by Darr and Fridovich. 14The ability of monovanadate to stimulate NADH oxidation both in the absence of exogenous source of O2- (Fig. 6b)

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h (n m) Fig. 4. Spectra of V,v) + V(v, mixtures in water or phosphate buffer. 2 mM V,v) was added to 2 mM V~v~ in water (1) or in 10 mM, 40 mM and 100 mM potassium phosphate buffers, pH 6.0 (2, 3, 5). After 5 min the spectra of the resulting blue-green compounds were recorded. The spectra of 2 mM V(tv> (4) and 2 mM V(v, (5) were recorded for comparison.

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Fig. 5. V ( i v ) - - V i v ) complex formation during V~v)-dependent NADH oxidation. 2 mM V(v) were added to 2 mM NADH in 100 mM HEPES buffer, pH 6.0 (line 1) or 100 mM potassium phosphate buffer, pH 6.0 (line 2).

V~v,-dependentNADH oxidation

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Fig. 6. UV Spectra and ability of monovanadate and decavanadate to catalyze NADH oxidation. (a)---UV spectra: 0.15 mM monovanadate (1); 0.15 mM decavanadate (2). (b)--Ability of monovanadateand decavanadate to catalyze NADH oxidation: 0.5 mM monovanadate(1) or 0.5 mM decavanadate(2) were added to 0.2 mM NADH in 100 mM potassium phosphate buffer, pH 6.0. and in the presence of Rose Bengal + light (data not shown) was much higher than that of decavanadate.

oxidation by a free radical mechanism described in detail elsewhere: 1°,~2,~3 V~vj + 02-

DISCUSSION This study at variance with the data of Vijaya and Ramasarma 17 suggests that the Vtv;dependent NADH oxidation in the absence of exogenous source of 02is a free radical chain process. The first stage of the V~v)-dependent NADH oxidation is the direct low-rate oxidation of NADH by Vtv) and the generation of Vtlv~. The Vtw) generated in a medium containing V~v~ and oxygen would either react with V~v) to form a V(v) V~v) complex relatively stable to reaction with oxygen (1) or autoxidize producing O2- (2):

NADH + VOO. + H ÷

NAD. + 02

V(w) + O2

> V(v) - V(rv)

(1)

) V(v) + 02-

(2)

Reaction (1) might actually occur in the absence of phosphate and reaction (2) in the presence of phosphate. This seems reasonable because phosphate prevented the formation of a V(v) - V(w) complex on mixing V(v) and V(lv) and on incubation of Vtv) with glucose or NADH, allowing the Vav ~autoxidation. The phosphate anion, which is structurally similar to vanadate, forms complexes with vanadyl. ~9 Competition between phosphate and vanadate anions for a complex formation with vanadyl should be expected. In the phosphate-vanadyl complex V(w) would be more easily oxidized by oxygen than V(w) in the vanadyl-vanadate complex. It follows that phosphate plays an important role in the V(v)-dependent NADH oxidation, favoring the O:- generation which, in turn, initiates NADH

(3)

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(5)

The fact that the Vtv)-dependent NADH oxidation is inhibited not only by SOD but also by catalase, mannitol and ethanol suggests the participation of the following reactions: V(w) + H202

V(v) + Vtw)

~ VOO.

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) V(v) + OH- + .OH

(6)

) H20 + NAD.

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In the presence of H202 V(iv) oxidizes NADH. This oxidation is strongly inhibited by ethanol and slightly by SOD. 16 The present finding of an almost equal rate of the Rose Bengal + light-catalyzed V(v)-dependent NADH oxidation in HEPES and phosphate buffers suggests that the free radical chain process, reactions (3)-(5), does not require phosphate. This is in agreement with the data of Darr and Fridovich, 14 who have reported that the V(v)-dependent NADH oxidation initiated by xanthine + xanthine oxidase-produced O2- is a phosphate-independent process. It is thus concluded that phosphate stimulates NADH oxidation only in the cases of vanadium-dependent O2- generation. Therefore, the absence or the presence of stimulation of the V(v)-dependent NADH oxidation by phosphate allows

354

S.I. LIOCHEVand E. A. IVANCHEVA

one to decide if a given system generates 02- in a vanadium-independent way, or reduces Vtv> to V~lv, which, in turn, generates O2-. Monovanadate was more active in producing NADH oxidation in the absence and in the presence of photosensitizers + light as compared to decavanadate. In other words, it was more active when 02- was generated in either a vanadium-dependent, or a vanadiumindependent way. Thus, our finding is in keeping with the data of Darr and Fridovich, ~4 who demonstrated that the effect of monovanadate was greater than that of decavanadate but are at odds with the results of other workers, 4-8'17 suggesting a higher activity of decavanadate as compared to that of monovanadate. The present study argues against the suggestion that the presumed decavanadate-phosphate complex, and not simply vanadate, is the active species causing the oxidation of NADH. 7's The V~vrdependent reactions described could lead to a depletion of NAD(P)H and an increased production of O2- in the cell. This finding, in agreement with other data of ours, 1°'15"16may partially explain the biological effects of vanadium. 18 Acknowledgment--This work was supported by the Committee for Science to the Council of Ministers.

REFERENCES 1. Erdmann, E.; Kraewietz, W.; Phillip, G.; Hackbarth, I.; Schmitz, W.; Scholz, H.; Crane, F. L. Purified cardial cell membranes with high (NA÷K +) ATP-ase activity contain significant NADH-vanadate reductase activity. Nature (London) 282:335-336; 1979. 2. Crane, F. L.; MacKellar, W. C.; Morre, D. J.; Ramasarma, T.; Goldenberg, H.; Grebing, C.; L6w, H. Adriamycin affects plasma membrane redox functions. Biochem. Biophys. Res. Commun. 93:746-754; 1980. 3. Menon, A. S.; Ran, M.; Ramasarma, T.; Crane, E L. Vanadate inhibits mevalonate synthesis and activates NADH oxidation in microsomes. FEBS Left. 114: ! 39-141 ; 1980 4. Ramasarma, T.; MacKellar, W. C.; Crane, F. L. Vanadatestimulated NADH oxidation in plasma membrane. Biochem. Biophys. Acta 646:88-98; 1981. 5. Vijaya, S.; Crane, F. L.; Ramasarma, T. A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide. Mol. Cell Biochera. 62:175-185; 1984.

6. Patole, M. S.; Kurup, C. K. R.: Ramasarma, T. Reduction of vanadate by a microsomal redox system. Biochem. Biophys. Res. Commun. 141:171-175; 1986. 7. Patole, M. S.; Kurup, C. K. R.; Ramasarma, T. NADH-dependent polyvanadate reduction by microsomes. Mol. Cell Biochem. 75:161-167; 1987. 8. Ran, M.; Patole, M. S.; Vijaya, S.; Kurup, C. K. R.; Ramasarma, T. Vanadate-stimulated NADH oxidation in microsomes. Mol. Cell Biochem. 75:151-159; 1987. 9. Briskin, D. P.; Thornley, W. R.; Poole, R. J. Vanadate-dependent NADH oxidation in microsomal membranes of Sugar beet. Arch. Biochem. Biophys. 236:228-237; 1985. 10. Liochev, S.; Fridovich, I. The vanadate-stimulated oxidation of NAD(P)H by biomembranes is a superoxide-initiated free radical chain reaction. Arch. Biochem. Biophys. 250:139-145: 1986. 11. Khadke, L.; Gullapall, S.; Patole, M. S.; Ramasarma, T. Vanadate-stimulated NADH oxidation by xanthine oxidase: An intrinsic property. Arch. Biochem. Biophys. 244:742-749; 1986. 12. Liochev, S.; Fridovich, I. Further studies of the mechanism of the enhancement of NADH oxidation by vanadate. J. Free Radical Biol. Med. 1:287-292; 1985. 13. Dart, D.; Fridovich, I. Vanadate and molybdate stimulate the oxidation of NADH by superoxide radical. Arch. Biochem. Biophys. 232:562-565; 1984. 14. Dart, D.; Fridovich, I. Vanadate enhancement of the oxidation of NADH by 02-: Effects of phosphate and chelating agents. Arch. Biochem. Biophys. 243:220-227; 1985. 15. Liochev, S.; Fridovich, I. The oxidation of NADH by vanadate + sugars. Biochem. Biophys. Acta 924:319-322; 1987. 16. Liochev, S.; Fridovich, I. The oxidation of NADH by tetravalent vanadium. Arch. Biochem. Biophys. 255:274-278; 1987. 17. Vijaya, S.; Ramasarma, T~ Vanadate stimulates oxidation of NADH to generate hydrogen peroxide. J. Inorg. Biochem 20:247-254; 1984. 18. Boyd, D. W.; Kustin, K. Vanadium: a versatile biological effector with an elusive biological function. Adv. lnorg. Biochem. 6:311-365; 1985. 19. Nechai, R. B.; Nanninga, L. B.; Nechai, P. S. E.; Post, R. L.: Grantham, J. J.; Macara, J. G.; Kubena, L. F.; Phillips, T. D.: Nielsen, F.H. Role of vanadium in biology. Fed. Proc. 45:123132; 1986.

ABBREVIATIONS

V~v,--vanadate V~iv~--vanadyl SODwCu,Zn-superoxide dismutase BHT--butylated hydroxytoluene O2---superoxide radical •OH--hydroxyl radical HEPES---N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid