Department of Pharmacology and Pharmacotherapy, University of Pécs, Medical School, Szigeti út 12, H-7624, Pécs, Hungary
Received April 5, 2002; accepted June 3, 2002
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ABSTRACT |
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Key Words: arsenate; arsenite; purine nucleoside phosphorylase; reduction; thiols.
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INTRODUCTION |
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The accompanying paper (Németi and Gregus, 2002) has characterized an AsV reductase activity in rat liver cytosol. This activity, which depended on the presence of an appropriate exogenous thiol compound, was enhanced up to 100-fold in the presence of 6-oxopurine nucleosides, i.e., inosine and guanosine, and was inhibited significantly by mercurial thiol reagents, inorganic phosphate, that is an analogue of AsV (Dixon, 1997
), as well as by 6-oxopurine nucleobases, i.e., hypoxanthine and guanine. These findings have prompted the tentative conclusions that the cytosolic AsV reductase may contain functionally important thiol group(s), may accept inosine or guanosine as well as inorganic phosphate as substrates and may yield hypoxanthine or guanine as products. The enzyme that fits the deducted characteristics is known as purine nucleoside phosphorylase (PNP).
PNP is a soluble enzyme localized in the cytosol and contains critical thiol groups (Parks and Agarwal, 1972; Bzowska et al., 2000
; Erion et al., 1997
). As shown in Figure 1
, this enzyme catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides, utilizing inorganic phosphate. PNP can cleave inosine to hypoxanthine, or guanosine to guanine, yielding, in either case, ribose-1-phosphate. This reaction is readily reversible; however, the forward reaction is favored in vivo because of rapid elimination of the products. Like many phosphate-utilizing enzymes, PNP also accepts AsV instead of phosphate and produces the purportedly unstable ribose-1-arsenate during arsenolysis of 6-oxopurine nucleosides (Kline and Schramm, 1993
).
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MATERIALS AND METHODS |
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Preparation of hepatic cytosol.
Cytosol from the liver of male Wistar rats (270330 g), CFLP mice (2835 g), English shorthair guinea pigs (450550 g), golden Syrian hamsters (90115 g), and New Zealand white rabbits (1.61.8 kg) was prepared by differential centrifugation of the 33% homogenate made in 150 mM KCl50 mM TRIS (pH 7.0), as described (Németi and Gregus, 2002). Cytosolic protein concentration was determined by the bicinchoninic acid method (Brown et al., 1989
). The cytosol was stored in 1-ml aliquots at 80°C until use. For anion exchange chromatography (see below), 500 µl cytosol (30 mg protein/ml) was desalted by centrifugal ultrafiltration at 4°C using a Microcon-30 filter. The retentate was washed twice with 20 mM TRIS (pH 7.6), after which the final retentate was taken up in 300 µl of 20 mM TRIS, pH 7.6.
Enzymatic assays.
For assaying AsV reductase activity, hepatic cytosol (5 mg protein/ml) or calf spleen PNP (50 mU/ml) was preincubated at 37°C for 5 min in 150 mM KCl50 mM TRIS (pH 7.6) containing a nucleoside (typically inosine) at a concentration indicated in the legends of figures and footnotes of tables. When the effect of inhibitor compounds was tested on AsV reductase activity, these compounds (except phosphate) were also present during preincubation, at concentrations specified (see Fig. 2 and Table 1
). Subsequently, the incubation was started by addition of a thiol compound (typically dithiothreitol, DTT), at a concentration specified in the legends, and AsV (25 µM). At 10 min after AsV addition, the reaction was stopped by addition of mersalyl (20 mM) to displace thiol-bound AsIII and, 15 seconds later, 3 volumes of ice-cold deoxygenated methanol to precipitate proteins. The methanolic incubates were then stored at 80°C until arsenic analysis. AsIII in the deproteinized methanolic incubates was separated from AsV and quantified by HPLC-HG-AFS, essentially as described for speciation of arsenic metabolites (Gregus et al., 2000
). In the present work, however, an isocratic elution, rather than gradient elution, was performed with 60 mM potassium phosphate (pH 5.75) at 1 ml/min flow rate.
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Anion exchange chromatography of cytosol.
Rat liver cytosol was fractionated using a Superformance 15010 glass column (Merck) filled with Fractogel EMD TMAE (S) anion-exchange resin to obtain a 100 x 10-mm gel column, 20 mM TRIS, pH 7.6 (eluent A) and 20 mM TRIS (pH 7.6)500 mM KCl (eluent B). The ice-cold eluents were pumped at a combined flow rate of 1 ml/min with 2 Waters-501 HPLC pumps operated under control of Millennium Chromatography Manager (Waters), through an injector (Rheodyne 7125) equipped with a 100-µl sample loop and then through the column, maintained at 4°C. After equilibrating the column by pumping eluent A for 30 min, 100 µl desalted cytosol (see above) was injected onto the column and eluted with 100% eluent A for 5 min. By 35 min, 100% eluent A was changed linearly to 100% eluent B, after which 100% eluent B was maintained for 10 min. During the 45-min elution, the eluate was collected in 1-ml fractions. The fractions stored on ice were assayed for PNP activity and AsV reductase activity within 3 h.
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RESULTS |
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The PNP inhibitors also markedly inhibited the DTT-supported AsV reductase activity of hepatic cytosol prepared from other species (Table 1). BCX-1777, at 1 µM concentration, practically abolished AsV reductase activity of rat, mouse, and hamster cytosols, whereas it decreased the activity to 2% and 13% of control, respectively, in the cytosols of guinea pigs and rabbits. At this concentration CI-1000 proved to be slightly less effective, lowering AsV reductase activities to 1, 4, 8, 15, and 14% of control, respectively, in the cytosols of rats, mice, guinea pigs, hamsters and rabbits (Table 1
).
Association between cytosolic AsV reductase and PNP activities.
We sought for association between cytosolic AsV reductase and PNP activities in 2 ways; first, AsV reductase activities in the hepatic cytosol of various species that were related to the PNP activities assayed in the same cytosols (Fig. 3). When AsV reductase activity was assayed without added inosine, a correlation appeared between these 2 activities in the hepatic cytosol of rats, guinea pigs, mice and rabbits, with decreasing PNP and AsV reductase activities in this order (Fig. 3
, left). However, the hamster, with its extremely low AsV reductase activity relative to its PNP activity, did not fit into this interspecies correlation. Nevertheless, when AsV reductase activity was assayed with added inosine, which necessitated the use of 50-fold diluted cytosols (Fig. 3
, right), the AsV reductase activity of the hamster rose much more than that of the other species. In this case also, only a rough interspecies correlation was observed, with the rabbit exhibiting the lowest PNP and lowest AsV reductase activities, whereas the other species with high PNP activities ranging from 115 to 225 nmol/min mg protein exhibited high, but comparable AsV reductase activities.
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DISCUSSION |
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Collectively, these observations constitute compelling evidence that the DTT-supported AsV reductase activity in the hepatic cytosol of rats, and other species tested here, is ascribable, depending on the species, completely or almost completely to PNP, because PNP can work as an AsV reductase and because specific PNP inhibitors completely or almost completely abolished the cytosolic AsV reductase activities in the species investigated. It appears most likely that the AsV reductase activity found recently in the liver of humans (Radabaugh and Aposhian, 2000) and nonhuman primates (Wildfang et al., 2001
) is also ascribable, at least in part, to PNP, because that AsV reductase was also supported by DTT (but barely by GSH) and required a heat-stable endogenous compound or compounds less than 3 kDa in size, which is likely to be inosine and/or guanosine, essential for the PNP-catalyzed AsV reduction.
PNP is a ubiquitous enzyme that cleaves purine nucleosides, thereby contributing to salvage of the released purine bases for reutilization in the synthesis of purine nucleotides (Bzowska et al., 2000; Parks and Agarwal, 1972
). This soluble enzyme consists of 3 identical subunits of approximately 32 kDa, contains 12 cysteines, and is sensitive to inactivation by p-chloromercuribenzoate (Parks and Agarwal, 1972
). Mammalian PNPs exhibit a very high degree of sequence homology and identical amino acids constitute the substrate binding sites in the human, bovine, rat, and mouse enzymes (Bzowska et al., 2000
; Erion et al., 1997
). PNP exhibits high substrate specificity for inosine and guanosine (6-oxopurines), whereas the 6-aminopurine adenosine is a very weak substrate (Bzowska et al., 2000
). For the second substrates phosphate and AsV, the human erythrocytic enzyme has KM values of 0.74 and 1.8 mM, respectively (De Verdier and Gould, 1963
).
The observations presented here indicate that PNP-catalyzed AsV reduction takes place during or as a consequence of the arsenolytic reaction, i.e., the forward reaction depicted in Figure 1. This conclusion is supported in part by the findings that AsV reduction is inhibited by phosphate that competes with AsV in the arsenolytic reaction (based on structural similarities of these oxyanions) and also by the observation that 6-oxopurine nucleoside substrates of PNP, which support the forward reaction, were required for PNP-catalyzed AsV reduction. Interestingly, adenosine, which is an extremely poor substrate for PNP, also supported to some extent the reduction of AsV by calf spleen PNP. This seemingly controversial finding is attributable to the fact that, according to the manufacturer, this enzyme preparation may contain up to 0.5% adenosine deaminase, which can convert the poor PNP substrate adenosine into the good substrate inosine. Furthermore, the observation that hypoxanthine and guanine, strong inhibitors of the phosphorolytic cleavage of purine nucleosides by PNP (Glantz and Lewis, 1978
; Parks and Agarwal, 1972
), strongly inhibited the PNP-catalyzed AsV reduction, also indicates that the forward reaction is essential for reduction of AsV to AsIII by this enzyme. It is unknown, however, whether the arsenolytic cleavage of 6-oxopurine nucleosides is a prerequisite for the PNP-catalyzed reduction of AsV, because 6-oxopurine nucleoside binding induces a favorable conformational change in the enzyme (Bzowska et al., 2000
; Mao et al., 1998
), and/or because, in this reaction, the AsV oxyanion becomes susceptible to reduction and/or it becomes accessible to the reducing moieties of the enzyme protein.
Regarding the mechanism of AsV reduction by PNP, views on the mechanism of AsV reduction by certain microbial AsV reductases may be enlightening. It has been proposed recently that the identical AsV reductases of Bacillus subtilis and Staphylococcus aureus work as a "triple cysteine redox relay" or a "disulfide cascade," using Cys-10, Cys-82, Cys-89, Arg-16, and Asp-105, as essential residues in the catalytic cycle, and the small dithiol protein, thioredoxin (Bennet et al., 2001; Zegers et al., 2001). Briefly, the reduction process is thought to be initiated by the nucleophilic attack of Cys-10 thiolate anion on AsV, after which AsV is reduced with the contribution of Cys-82 thiolate, resulting in formation of AsIII and a disulfide bond between these cysteines. This disulfide bond will then be broken by Cys-89, with recovery of Cys-10 thiolate group and formation of a new disulfide bond between Cys-82 and Cys-89, which in turn are reduced by thioredoxin, thus reactivating the enzyme. Importantly, Cys-10, which initiates AsV reduction by this bacterial AsV reductase, is part of a CX5R signature motif (in which C and R represent Cys-10 and Arg-16), which is also found in the phosphate-binding loop of low molecular weight protein tyrosine phosphatases (Bennett et al., 2001
; Zegers et al., 2001
). The CX5R motif is also present in the AsV reductase of Saccharomyces cerevisiae and is essential for its catalytic function (Mukhopadhyay and Rosen, 2001
). It is important to point out that the human, bovine, rat, and mouse PNPs also contain one CX5R motif in each subunit, with Cys-78 and Arg-84 representing C and R in this motif (Bzowska et al., 2000
; Erion et al., 1997
). Moreover, Arg-84 is involved in the phosphate (and AsV) binding site of PNP (Mao et al., 1998
; Bzowska et al., 2000
). Therefore, it is tempting to hypothesize that the CX5R motif represents the catalytic center of PNP when functioning as an AsV reductase, just as it does in the above-mentioned microbial AsV reductases. It remains to be analyzed whether this hypothesis is verifiable, whether other cysteines in PNP can assume the same role as Cys-82 and Cys-89 can in bacterial AsV reductase (see above), and whether DTT and some other thiols assume the function of thioredoxin (or some other endogenous thiols) in returning the protein into an active form capable of reducing AsV.
In summary, this paper demonstrates that PNP can fortuitously function as an AsV reductase when catalyzing the arsenolytic cleavage of inosine or guanosine in the presence of an appropriate thiol. Furthermore, the findings of this and the accompanying work (Németi and Gregus, 2002) indicate that it is the PNP that is largely or exclusively responsible for the thiol-dependent reduction of AsV to AsIII in the hepatic cytosol of rats, mice, hamsters, guinea pigs, and rabbits. Further research is warranted to clarify the mechanism of PNP-catalyzed AsV reduction and the in vivo role of PNP in forming the toxic AsIII from the less toxic and environmentally prevalent AsV.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: 36-72-536-218. E-mail: zoltan.gregus{at}aok.pte.hu.
Note added in proof: After acceptance of this work for publication, it was reported (Radabaugh, T. R., Sampayo-Reyes, A., Zakharyan, R. A., and Aposhian, H. V. [2002]. Arsenate reductase II. Purine nucleoside phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems. Chem. Res. Toxicol. 15, 692698) that the purified human liver arsenate reductase characterized earlier by Radabaugh et al. (2000) has an amino acid sequence identical with that of human purine nucleoside phosphorylase.
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