Vanadate-mediated oxidation of NADH: Description of an in vitro system requiring ascorbate and phosphate

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 272, No. 1, July, pp. 76-80,1989

Vanadate-Mediated SHIN

Oxidation of NADH: Description of an in Vitro System Requiring Ascorbate and Phosphate

YOSHINO,

Department

STEPHEN

of Pharmacology, Received

New

September

GENE

York

SULLIVAN,

University

12,1988,

School

and in revised

AND

of Medicine, form

February

ARNOLD New

York,

STERN’ New

York

10016

20,1989

Oxidation of NADH has been observed in an in vitro system requiring NADH, vanadate, ascorbate, and phosphate. Similar results were observed with NADPH. Ascorbate provides the reducing equivalents necessary to reduce vanadate to vanadyl. Vanadyl autoxidizes producing superoxide which initiates a free radical chain reaction resulting in oxidation of NADH. Oxidation is inhibited by superoxide dismutase but not by catalase or ethanol. Ascorbate functions to initiate the free radical chain reaction but is not required in stoichiometric concentrations. At higher concentrations, ascorbate inhibits NADH oxidation. Inorganic phosphate was required for NADH oxidation. Dialysis of phosphate buffers against solutions containing apoferritin or conalbumin or addition of transition metal cations or chelators to the reaction medium did not alter dependence on phosphate. Phosphate and vanadate were interchangeable in their effects on kinetic parameters of NADH oxidation except that vanadate was 100 times more potent than phosphate. Vanadate participates directly in the initiating and propagating redox reactions of NADH oxidation. Phosphate may be important in lowering the energy of activation for the necessary transfer of hydronium ion and water in the transition state between vanadate anion and vanadyl cation. o 1989 Academic press, I,,~.

showed that ascorbate can efficiently reduce vanadate to vanadyl. We have recently investigated the effect of ascorbate on reduced pyridine nucleotide oxidation (7). We have found that vanadate plus ascorbate produces a rapid oxidation of NAD(P)H which was inhibited by superoxide dismutase. We have also observed that the oxidation of reduced pyridine nucleotides is dependent on the phosphate concentration.

Vanadate has been reported to catalyze the oxidation of NAD(P)H in the presence of subcellular systems prepared from mouse liver plasma membrane and erythrocyte membrane (1,2) and rat liver microsomes (3). In these systems exogeneous chemical and enzymatic sources of reducing equivalents are required for initiation of free radical chain reactions. Recent studies have shown that vanadyl, the reduced form of vanadate, autoxidizes with the production of 0, and vanadate, followed by NAD(P)H oxidation (4). No exogenous source of reducing equivalents was required for this oxidation. Liochev and Fridovich (5) have demonstrated that sugars and sugar phosphates can reduce vanadate to vanadyl enabling vanadate to catalyze NADH oxidation. Adam-Vizi et al. (6) 1 To whom 0003-9861189 Copyright All rights

correspondence

should

$3.00

Q 1989 by Academic Press, Inc. of reproduction in any form reserved.

MATERIALS

AND

METHODS

Sodium orthovanadate was purchased from Aldrich Chemical Co. (Milwaukee, WI). Reduced pyridine nucleotides, sodium ascorbate, superoxide dismutase, and catalase were supplied from Sigma (St. Louis, MO). All other reagents were of reagent grade quality. For the measurement of NAD(P)H oxidation, 10 ~1 of sodium orthovanadate dissolved in water was mixed with 980 ~1 of NkHP04, pH 7.4 (HCI), containing NAD(P)H in the spectrophotometric cuvette and

be addressed. 76

VANADATE-MEDIATED TABLE EFFECTS OF ASCORBATE

OXIDATION

I

CONCENTRATION

ON NADH

OXIDATIONINTHEPRESENCE OFVANADIUM

@ADHI bM)

[Ascorbate]

Cm@ 0 0.0005 0.005 0.05 0.1 0.5

0.100 0.069 0.013 0.007 0.011 0.027

-

-

Note. The concentration of NADH after incubation at 25°C for 1 h was measured by the absorbance at 340 nm. Initial concentrations were 100 mM NasHPOd, pH 7.4 (HCI), and 0.5 mM sodium orthovanadate.

then 10 ~1 of ascorbate dissolved in water was added and mixed. Final concentrations were 0.5 mM sodium orthovanadate, 0.1 mM NAD(P)H, 0.05 mM sodium ascorbate, 100 mM NazHPOl, and 1% (v/v) ethanol. In some experiments, varying concentrations of sodium orthovanadate, ascorbate, and phosphate were used under the same conditions. Five micrograms of superoxide dismutase or catalase in a small volume of water was added to the reaction mixtures before or after the addition of ascorbate. Ethanol was added before the addition of ascorbate. All reactions were carried out at 25°C. The decrease in absorbance at 340 nm due to NAD(P)H oxidation was measured. RESULTS

NADH oxidation can be induced by vanadate anion (VO;) but only in the presence of a source of reducing equivalents to generate catalytic amounts of vanadyl cation (VO”) (5). Ascorbate can serve as the source of reducing equivalents as shown in Table I. Maximum NADH oxidation occurs at 0.05 mM ascorbate. Higher ascorbate concentrations inhibit NADH oxidation. Significant NADH oxidation (0.087 mM in 1 h) occurs with as little as 0.005 mM ascorbate. The oxidation of NADPH in the presence of vanadate and ascorbate was similar to that shown for NADH (data not shown). We have previously shown that inorganic phosphate is required for NADH oxidation in the ascorbate-vanadate system (7). Trace metal contamination of the

OF

NADH

77

phosphate buffer was tested by dialyzing the buffer against solutions containing apoferritin or conalbumin to remove trace metals with no resulting effect on phosphate dependency of NAD(P)H oxidation. There was also no effect of metal chelating agents, desferrioxamine, or diethylenetriaminepentaacetic acid on NAD(P)H oxidation. The addition of up to 10 pM Fe’+, Cu’+, or Zn2+ to the phosphate buffer had no effect on oxidation of NAD(P)H. NADH oxidation in the presence of ascorbate, vanadate, and phosphate has been shown to accelerate to a maximum rate after a lag phase (7). Increasing the phosphate concentration decreases the lag phase and increases the maximum oxidation rate (7). Holding the NADH, ascorbate, and phosphate concentrations constant while varying the vanadate concentration shows that vanadate is similar to phosphate in decreasing the lag phase and increasing the maximum oxidation rate with increasing vanadate concentrations (Fig. 1). Experiments were then carried out to create tables in which the appropriate combination of vanadate and phosphate concentrations could be selected for a desired lag phase (Table II) and maximum rate (Table III) of NADH oxidation. Although vanadate and phosphate are both required for NADH oxidation in our system, vanadate is approximately 100 times as potent as phosphate.

FIG. 1. Effect of vanadate concentration on NADH oxidation. Reaction mixtures contained 100 mM Na2HP04, pH 7.4 (HCl), 0.1 mM NADH, and 0.05 mM sodium ascorbate. Sodium orthovanadate was present at; a, 0 mM; b, 0.05 mM; c, 0.1 mM; d, 0.2 mM; or e, 0.5 mM. Reactions were carried out at 25°C.

78

YOSHINO, SULLIVAN, TABLE II

TIME ELAPSED TO REACH MAXIMUM RATES OF NADH OXIDATION AT VARIOUS VANADATE AND PHOSPHATE CONCENTRATIONS [Vanadate]

0

0.05

(mM)

0.1

0.2

0.5

142 113 71 2’7

116 80 28 9

Time (min) 0 12.5 25 50 100

-

129 98

136 106 69

AND STERN

idizes NADH. They also showed that sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH by reducing vanadate to vanadyl (5). Vanadylinduced oxidation of NADH was inhibited by superoxide dismutase but not by ethanol, a scavenger of OH’ (5). No exogeneous source of 0, was required under these conditions. Therefore, they suggested that vanadyl autoxidized producing 0, plus vanadate (reaction [Z]), followed by the oxidation of NADH by a free radical chain reaction (formalized by reactions [3] and [4]): V02+ + O2 + 2HzO + VO, + 0, + 4H+ [2]

Note. The time elapsed to reach maximum rates of NADH oxidation was measured as the time from the beginning of addition of ascorbate to the reaction mixture to the inflection point (see Fig. 1) at each combination of vanadate and phosphate concentrations. Initial concentrations were 0.1 mM NADH and 0.05 mM sodium ascorbate. Incubations were carried out in Na,HPO,, pH 7.4 (HCI), at 25OC.(-) A time greater than 180 min.

The effects of O,, Hz@, and OK on NADH oxidation were studied by the addition ofsuperoxide dismutase, catalase, and ethanol, respectively (Fig. 2). Superoxide dismutase inhibited NADH oxidation whether added at zero time or at 9 min. Catalase and ethanol had no effect.

NADH + 0, + I-I+ + NAD’ + Hz02

I31

NAD’ + Oz 3 NAD+ + 0;

C4J

In our studies the oxidation of the reduced nucleotides by vanadate plus ascorbate was also inhibited by superoxide dismutase, but not by catalase or ethanol, supporting their suggestion. Reduced ascorbate is not stoichiometric in driving a cooxidation of NADH plus ascorbate but rather initiates a free radical chain reaction. This is shown by the

TABLE III MAXIMUM RATES OF NADH OXIDATION AT VARIOUS VANADATE AND PHOSPHATE CONCENTRATIONS

DISCUSSION

[Vanadate] (mM)

The present studies show that rapid NAD(P)H oxidation by vanadate is induced in the presence of ascorbate. The optimum dose of ascorbate is similar to the concentration in biological tissues. There was little oxidation of NAD(P)H by vanadate itself. This indicates that the oxidation of the reduced pyridine nucleotides was induced by reduction of vanadate (VO;) to vanadyl (VO”‘) by ascorbate (AH-): VO; + AH- + 3H+ + VO’+ + A: + 2HZ0

0

Our results support the previous finding by Liochev and Fridovich (4) that vanadyl ox-

0.1

NADH oxidation 0 12.5 25 50 100

------

0.84 0.94

0.2 (&tM

0.94 1.08 1.44

0.5

NADH/min) 0.98 1.08 1.54 7.28

1.16 1.68 8.96 27.3

Note. Maximum rates of NADH oxidation were measured after the lag period and at the inflection point (see Fig. 1) for each combination of vanadate and phosphate

[l]

0.05

concentrations.

Initial

concentrations

were 0.1 rnM NADH and 0.05 mM sodium ascorbate. Incubations were carried out in NeHPO,, pH 7.4 (HCI), at 25°C. (-) A rate Iess than 0.05 @f NADH/ min.

VANADATE-MEDIATED

OXIDATION

OF NADH

79

in red cells by vanadate. There was also no oxidation of NAD(P)H in red cells preincubated with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Vanadate was shown to cross the red cell plasma membrane by an anion-exchange system (9,10) and to interact with red cell metabolism (11). Vanadate was also reported to catalyze NAD(P)H oxidation in the presence of mouse liver plasma membrane and erythrocyte membrane (1,2) and rat liver microsomes (3). Therefore, our results in FIG. 2. Effect of superoxide dismutase, catalase, or the cellular system suggest that there is an ethanol on NADH oxidation. Reaction mixtures contained 100 mM NaaHPO,, pH 7.4 (HCI), 0.1 mM NADH, intracellular inhibitory mechanism(s) of 0.5 mM sodium orthovanadate, and 0.05 mM sodium NAD(P)H oxidation by vanadate plus ascorbate. Additions: a, none; b, 5 pg/ml superoxide ascorbate in red cells. The present studies dismutase at 0 min; c, 5 pg/ml superoxide dismutase show that the oxidation of reduced pyriat 9 min; d, 5 pg/ml eatalase at 0 min; e, 1% (v/v) dine nucleotides requires a high concentraethanol at 0 min. Reactions were carried out at 25°C. tion of sodium phosphate. A concentration greater than 50 mM Na2HP04 buffer was necessary for efficient NAD(P)H oxidafact that 0.005 mM ascorbate allows oxidation. The lack of oxidation of NAD(P)H in tion of 0.087 mM NADH in 1 h. Ascorbate the red cell may result from the relatively at concentrations greater than 0.05 mM in- low intracellular concentration of phoshibited NADH oxidation. It can be anticiphate. For example, in Krebs-Ringer pated that at these higher concentrations, phosphate buffer (16.5 mM Na2HP04) or reduced ascorbate would reduce the sodium chloride, no significant oxidation of steady-state concentration of superoxide NAD(P)H by vanadate plus ascorbate occurred. The addition of excess NazHPO, (8): to Krebs-Ringer phosphate buffer or AH-+O,+H++A-+H,O, [51 sodium chloride caused an enhancement of Ramasarma and colleagues (1, 2) have NAD(P)H oxidation (7). noted that when orthovanadate is disA question may arise as to the possibility of the involvement of trace metals in solved in phosphate buffer or acidified with HCl, or when vanadium pentoxide is dis- the NAD(P)H oxidation which may be solved in NaOH, the resulting solutions present in phosphate buffer. However, this is unlikely since significant NADH oxidatake on a yellow color. This color is probably due to the higher percentage of deca- tion by vanadate plus ascorbate was also vanadate, a polymeric vanadium oxide an- induced in phosphate buffer which was diion, in these solutions. These yellow solualyzed against a solution containing apotions showed higher vanadate-stimulated ferritin or conalbumin which could remove NADH oxidation in their systems (1, 2). trace metals in the buffer. There was also Yellow solutions prepared by acidification no effect of metal chelating agents, desferof orthovanadate solutions showed no difrioxamine, and diethylenetriaminepentaferences in the ascorbate-induced NADH acetic acid on the NAD(P)H oxidation. The oxidation system (data not shown). addition of 1 to 10 I.LM Fe(U), Cu(II), or Because the present studies showed a Zn(I1) to the phosphate buffer showed litrapid oxidation of NAD(P)H by vanadate tle effect on the oxidation of the reduced plus ascorbate, we have recently tested pyridine nucleotides. These results show whether NAD(P)H in the intact red blood that the NAD(P)H oxidation by vanadate cell incubated with vanadate is oxidized plus ascorbate is dependent on phosphate. The length of the lag period before maxi(7). We found that little oxidation of the reduced pyridine nucleotides was induced mum rates of NADH oxidation is reached

80

YOSHINO,

SULLIVAN,

is most likely dependent on the rate at which adequate concentrations of catalytic intermediates are achieved. Candidates for such key catalytic intermediates are VOzf, A:, O;, and NAD’. The dependence of the lag period and maximum rate of NADH oxidation on phosphate concentration implies that phosphate may be important in forming complexes with precursors of these intermediates. Of interest in this regard is that when V02+ is added to NADH solutions (as vanadyl sulfate), oxidation of NADH occurs in the absence of ascorbate or phosphate (data not shown). In particular, phosphate may be important in lowering the energy of activation for the necessary transfer of hydronium ion and water in the transition state between VO, and V02+ (reactions [l] and [2]). Vanadate and phosphate behave similarly in this in vitro NADH oxidation system except that vanadate is 100 times more potent than phosphate. Phosphate may mimic vanadate in many of its physical and chemical properties (12-14). Although phosphate cannot participate directly in the initiating and propagating redox reactions for which vanadate is required, phosphate and vanadate were otherwise interchangeable. Desired kinetic parameters for NADH oxidation could be obtained by selecting any concentrations from a family of paired vanadate and phosphate concentrations. Vanadate has been shown to be present in most animal tissues (12) but its biological role is unknown (13). Vanadate’s effects on ATPase activity (9), insulin activity (15, 16), and metabolism of organic phosphates (11,14,17) may all be related to the ability of vanadate to substitute for phosphate at binding sites. Studies of vanadate-mediated oxidation of NAD(P)H have been carried out in phosphate buffers (l-4). Our results indicate that interaction of phos-

AND STERN

phate and vanadate may be important in vanadate-mediated NAD(P)H oxidation. ACKNOWLEDGMENT This work was supported by Grant ES03425 from the National Institutes of Health. REFERENCES 1. RAMASARMA,

T., MACKELLAR, W. C., AND CRANE, F. L. (1981) Biochim Biophys. Acta 646,88-98. 2. VIJAYA, S., CRANE, F. L., AND RAMASARMA, T. (1984) Mol. Cell. Biochem 62,175-185. 3. LIOCHEV, S., AND FRIDOVICH, I. (1986) Arch. B+ them. Biophys. 250,139-145. 4. LIOCHEV, S., AND FRIDOVICH, I. (1987) Arch Biochenz. Biophys. 255,274-278. 5. LIOCHEV, S., AND FRIDOVICH, I. (1987) Biochim Biophys. Ada 924,319-322. 6. ADAM-VIZI, V., VARADI, G., AND SIMON, P. (1981) J. Neurochem 36,1616-1620. 7. YOSHINO, S., SULLIVAN, S. G., AND STERN, A.

(1989) in Medical, Biochemical and Chemical Aspects of Free Radicals, Elsevier Science, Amsterdam, in press. 8. SULLIVAN, S. G., AND STERN, A. (1981) Biochem Ph.a?w4KoL 30,2279-2285. 9. CANTLEY, L. C., AND AISEN, P. (1979) J. BioL Chem 254,1781-1784. 10. HEINZ, A., RUBINSON, K. A., AND GRANTHAM, J. J. (1982) J. Lab. Clin. Med 100,593-612. 11. NINFALI, P., ACCORI, A., FAZI, A., PALMA, F., AND FORNAINI, G. (1983) Arch. Biochem. Biophys.

226,441-447. 12. SIMONS, T. J. B. (1979) Nature @und.m) 281,337338. 13. MACARA, I. G. (1980) !i”rends Biochm Sci 5,9294. 14. CARRERAS, M., BASSOLS, A. M., CARRERAS, J., AND CLIMENT, F. (1988) Arch Biochem Biophys.

264,155-159. 15. SHECHTER, Y., AMIR,

S., AND MEYEROVITCH, J. (1988) Diabetes Nutr. Metub. l, l-5. 16. TOROSSIAN, K., FREEDMAN, D., AND FANTUS, I. G. (1988) J. BioL Chem 263,9353-9359. 17. BENABE, J. E., ECHEGOYEN, L. A., PASTRANA, B., AND MARTINEZ-MALDONADO, M. (1987) J. BioL

Chem 262,9555-9560.