Purification and some properties of xanthine dehydrogenase from wheat leaves

Plant Science 134 (1998) 89 – 102

Purification and some properties of xanthine dehydrogenase from wheat leaves Paolo Montalbini * Istituto di Patologia Vegetale, Uni6ersita` di Perugia, Borgo XX Giugno 74, 06100 Perugia, Italy Received 27 November 1997; received in revised form 23 January 1998; accepted 24 February 1998

Abstract Wheat leaf xanthine oxidoreductase was purified to electrophoretic homogeneity. Due to the low activity in leaves, 0.5 mg of pure protein with 1–3 nkat activity was obtained from 1 kg of leaf material at the end of purification. Based on uric acid production, the pure enzyme specific activity was 1.8 and 6.5 nkat mg − 1 with NAD + and PMS as acceptors, respectively. Molecular oxygen alone did not accomplish substrate hydroxylation, indicating that the wheat leaf xanthine oxidizing enzyme is strictly a xanthine dehydrogenase (XDH) (EC 1.1.1.204., formerly EC 1.2.1.37.). The total ineffectiveness of wheat leaf XDH to utilize NADP + , FAD and ferricyanide as oxidizing substrates, the inhibition of NAD + reduction under anaerobiosis and the atypical visible absorption spectrum characteristics (maximum at 329–332 nm, a shoulder at about 392 – 394 nm and low absorption peak from 480 to 510 nm) differentiate wheat leaf XDH from other similar enzymes. Inhibitor effects, particularly p-hydroxymercuribenzoate and salicylhydroxamic acid, are instead in agreement with those reported for other xanthine oxidases and dehydrogenases. Wheat leaf XDH affinity for reducing substrates was very high with Km values of 4 – 8 mM. Oxidizing substrates instead showed a rather high Km value for NAD + but much lower for PMS and NBT. Wheat leaf XDH is inactivated by incubation of the enzyme with the physiological electron donors xanthine and hypoxanthine but this effect was reversed by a prolonged reaction time. Although the fluorescence spectrum emission maximum was at 329–332 nm indicating the presence of flavin in wheat leaf XDH molecule, the exact nature of prosthetic groups remains to be verified. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Xanthine dehydrogenase; Purification; Characteristics; Triticum aesti6um

1. Introduction Abbre6iations: DCPIP, dichlorophenol indophenol; NAD + , nicotinamide adenine dinucleotide; NBT, nitrobluetetrazolium; PMS, phenazine methosulfate. * Tel.: + 39 75 5856464; fax: +39 75 5856482; e-mail: [email protected]

Molybdenum iron-sulfur flavin hydroxylases are widely distributed enzymes (for pertinent literature see [1]) which includes two xanthine-oxidiz-

0168-9452/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0168-9452(98)00046-6

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P. Montalbini / Plant Science 134 (1998) 89–102

ing enzymes (xanthine oxidase: EC 1.2.3.2; and xanthine dehydrogenase: EC 1.1.1.204., formerly EC 1.2.1.37.) with a common multiple redox center constituted by FAD and Mo-pterine (two electron acceptors), and two Fe2S2 iron-sulfur clusters (one-electron acceptors) as prosthetic groups [2–4]. The latter specifically oxidize both xanthine and hypoxanthine and allopurinol which is also a potent inhibitor [5]. Xanthine oxidase (type O) is so called because of its efficient use of O2 as an electron acceptor in the hydroxylation of xanthine to uric acid; one of the most studied xanthine oxidases is the enzyme present in milk but many mammalian species and bacteria are also sources of this enzyme. In contrast, xanthine dehydrogenase (type D), which is present in avian (chicken and turkey) and mammalian (rat) liver, catalyzes the hydroxylation of xanthine using NAD + as the electron acceptor. Mammalian xanthine oxidases including the milk enzyme are known to be NAD + -dependent in freshly prepared extracts but during isolation and storage or with artificial treatment, such as thiol oxidation and/or proteolysis, the NAD + -dependent type is (reversibly or irreversibly) converted to the O2-dependent type [5]. This aspect is even more intriguing if we consider that crude or purified milk enzyme which behaves as an oxidase can be converted [6] or prepared and purified [7] in a dehydrogenase form in the presence of dithiothreitol. It now seems established that the two forms of the enzyme have different environments around their prosthetic flavin group [7,8] and that conformational changes in this environment create the condition for a binding site for NAD + . However, contrary to mammalian enzymes, the chicken liver enzyme has never been converted to the O2-dependent type indicating a very stable catalytic function in the FAD environment of this enzyme probably connected with a different nature of the protein structure. Investigations of unicellular photosynthetic organisms has been limited to the detection, isolation, purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii [9,10], while in higher plants dehydro-

genase enzymes were identified from a variety of plant leaf tissues [11,12] as well as in bean [13] and soybean [14,15] nodules. In all cases these enzymes showed considerable activity with NAD + and other artificial oxidants but did not react with oxygen at a significant level. Molecular properties of the above mentioned enzymes are limited. Purification to homogeneity was obtained in Chlamydomonas [10] and soybean nodules [14]. These two enzymes are molybdoflavoproteins but in the soybean nodule the flavin cofactor is FMN rather than FAD. All the studies on xanthine dehydrogenase from higher plants include information of Mendel and Mu¨ller [16] who detected the enzyme in a callus culture extract of Nicotiana tabacum, and many reports by Nguyen [17] which mainly deal with dark activation of leaf enzyme utilizing either an in vivo assay or assays of crude extracts. During studies on the involvement of purine catabolism enzymes in host-pathogen relationships, a xanthine oxidoreductase activity was revealed and quantified in crude leaf extracts from different plant sources after enzyme immobilization on polyacrylamide gel rods following electrophoresis [18–21]. Superoxide production by these leaf xanthine oxidoreductases was deduced indirectly from the inhibition by superoxide dismutase of the reduction of some artificial acceptors (nitrobluetetrazolium and phenazine methosulfate) used to oxidize hypoxanthine. This inhibitory effect was less pronounced [19–21] in wheat leaf xanthine oxidoreductase compared to bean and tobacco (about five times more superoxide dismutase was needed to produce a 50% inhibition of acceptor reduction with wheat enzyme). Thus, previous experimental evidence prompted to undertake a study to clarify the nature of wheat leaf oxidoreductase. The present data concern purification to electrophoretic homogeneity of this enzyme and some biochemical properties indicating that wheat leaf xanthine oxidizing enzyme is strictly a xanthine dehydrogenase with no possibility for a hydroxylation reaction of the substrate when oxygen is the only acceptor in the reaction mixture.

P. Montalbini / Plant Science 134 (1998) 89–102

2. Materials and methods

2.1. Plant material and growing conditions Wheat (Triticum aesti6um L.) cvs Mentana, Aurelio and Mirtos plants were grown during September–November under natural field conditions in two subsequent years (1995 – 1996). About 1 – 2 months from planting (5 – 6 leaf stage) leaf material was collected and kept frozen in plastic bags at − 25°C for about 1 month (and up to 1 year) before used for enzyme purification.

2.2. Enzyme assay All enzyme assays were performed aerobically at room temperature unless otherwise stated. Xanthine dehydrogenase activity was assayed: (i) by determination of uric acid production from xanthine by HPLC; (ii) following nicotinamide adenine dinucleotide (NAD + ) reduction spectrophotometrically at 340 nm; (iii) by polyacrylamide gel electrophoresis according to Mendel and Mu¨ller [16] with modifications using hypoxanthine as substrate and nitrobluetetrazolium as acceptor to detect the enzyme in all fractions during purification, particularly in crude extracts. Activity was routinely measured in an assay mixture (1.2 ml) of 0.13 M sodium pyrophosphate (NaPPi) buffer (pH 8.0) containing 1 mM xanthine or hypoxanthine, 1 mM NAD + or 0.1 mM phenazine methosulphate (PMS) and enzymatic solution (1–2 mg of pure preparation). Nitrobluetetrazolium (NBT) reduction was determined spectrophotometrically following the increase in absorbance at 540 nm. In experiments performed under anaerobiosis, the reaction mixture was purged by bubbling pure helium for 15 min before and 10 min after addition of the enzyme to a sealed spectrophotometric cuvette.

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supernatant (about 1.4 ml) was then applied to 3-ml Supelclean LC-18 tubes (Supelco, Inc. R&D Manufacturing Facility Bellefonte, PA) and uric acid freed of most acceptors and other pigments (especially with crude extracts) by elution with 1.5 ml of 0.1% acetic acid and 5% acetonitrile water solution. Separation and quantification of uric acid was then performed by HPLC chromatography using a reverse-phase column (120× 4 mm I.D.) packed with C18 particles (Hypersil ODS C18 5 mm, Sa¨ulentechnik, Dr Ing. H. Knauer GmbH, Am Schlangengraben 16, D-13597 Berlin) and a Perkin-Elmer HPLC system (Perkin-Elmer Corporation, 761 Main Ave., Norwalk, CT 06859-0012) with a 200 LC dual-pump, variablewavelength LC-75 ultraviolet programmable detector at 292.5 nm, and a LCI-100 computing integrator. Separation of uric acid was obtained by isocratic elution with 0.1% acetic acid in water at 0.8 ml min − 1. Uric acid peak identification was based on retention time, coelution with pure standard and enzymatic transformation by uricase (porcine liver uricase obtained from Sigma (St. Louis, MO) treatment. A typical elution pattern is reported in Fig. 1.

2.4. Electrophoresis Non-denaturing gel electrophoresis was performed in 7% polyacrylamide gel rods without stacking gel and protein sample applied in 20% glycerol. The optimal electrophoretic conditions were obtained using a continuous system where the same 50 mM Tris-boric acid buffer and 2.5 mM EDTA at pH 8.5 was used in the resolving gel and in the upper and lower electrode chambers. Analytical electrophoresis was run at 5 mA per gel for about 1 h until bromophenol blue tracking dye reached the bottom of the gel column.

2.3. HPLC analysis 2.5. Purification procedure After the reaction perchloric acid at a final concentration of 0.5 M was added to the assay mixture. After about 1 min the solution was neutralized with 4 M KOH solution and the insoluble perchlorate removed by centrifugation. The clear

Enzyme activity was measured by the described methods. Both NAD + and phenazine methosulphate (PMS) were used as oxidizing substrates for uric acid production from xanthine.

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P. Montalbini / Plant Science 134 (1998) 89–102

2.5.1. Preparation of crude homogenate and heat treatment Frozen wheat leaves, about 1 kg, were homogenized in a 1-gallon Waring Blender (Model CB-5, Waring Product Co., Winsted, CN) with

3 ml g − 1 of 0.1 M phosphate buffer, pH 7.8, containing 2 mM EDTA, 20 mM 2-mercaptoethanol and 0.5 mM sodium molybdate. The blender was run at full speed for 2 min and the filtered homogenate placed in a water bath maintained at 70°C, stirred for about half an hour and brought to 60°C. The heated solution was then cooled to 10°C and the precipitate removed by centrifugation at 10000× g and 5°C for 15 min.

2.5.2. Acetone fractionation Cold acetone (− 20°C) was stirred into to the clear supernatant to reach 40% (v/v). The precipitate was removed by centrifugation at 5000×g (5 min at 4°C). Acetone was added to the supernatant fluid to a final concentration of 62% (v/v). The precipitate was collected by centrifugation and dissolved in a minimal volume of the extraction buffer and then dialyzed overnight in the cold room against 100 vol of 0.01 M phosphate buffer, pH 7.8, 0.5 mM EDTA, 2 mM 2-mercaptoethanol and 0.2 mM sodium molybdate. 2.5.3. Protamine sulfate treatment To the dialyzed solution, clarified by centrifugation, a 2% solution of protamine sulfate in water was added dropwise to obtain 0.33 mg protamine mg − 1 protein in the enzymatic solution. The inactive heavy precipitate was removed by centrifugation.

Fig. 1. Separation and quantification of uric acid produced from xanthine by HPLC using a reverse-phase column (120 × 4 mm I.D.) packed with C18 particles (Hypersil ODS C18 5 mm). Two peaks probably corresponding to the enol and keto forms of uric acid were resolved, both sensitive to uricase treatment. The number on the top of peaks are the retention times in min. Only one peak in commercial uric acid used as standard with a retention time of 4.48 min (left), two peaks of uric acid produced from xanthine in the XDH-catalyzed reaction in our reaction conditions with 3.96 and 4.63 min of retention time, respectively (middle), and disappearance of the two peaks after uricase treatment (right).

2.5.4. Chromatography on DEAE-Cellulose The clear supernatant was loaded to a microgranular DEAE-cellulose (DE 52, Whatman, Whatman Ltd, Springfield Mill, Maidstone, Kent, UK) column (5×10 cm) previously equilibrated with the dialysis buffer, washed with 100 ml of the same buffer and 50 ml containing 50 mM NaCl. Variable amounts of activity was recovered in the unbound fraction. The column was then developed by two-step elution with 150 ml buffer, 150 and 300 mM in NaCl, respectively. Variable amounts of activity were recovered for these eluates for different leaf samples collected at different times.

P. Montalbini / Plant Science 134 (1998) 89–102

2.5.5. First ammonium sulfate fractionation The DEAE-cellulose unbound fraction and the bound fractions (eluted with 150 or 300 mM NaCl) were separately pooled and then subjected to ammonium sulfate fractionation. Solid ammonium sulfate was added slowly and dissolved to give 28% saturation and the pelleted inactive precipitate discarded. The supernatant was brought to 38% saturation and the resulting precipitate recovered by centrifugation and dissolved in a minimal volume of 25 mM Tris-boric acid at pH 8.5, 2.5 mM EDTA, 2 mM 2-mercaptoethanol and 0.2 mM sodium molybdate (TBEMM) and then dialyzed against the same buffer. 2.5.6. Gel filtration The clear supernatant was then filtered through a column (2.5×70 cm) of Sephacryl S-300 HR (Pharmacia, Biotech, Uppsala, Sweden) equilibrated with TBEMM buffer. Chromatography was carried out overnight in the cold room at a flow rate of 0.3 ml min − 1 and 5-ml fractions were collected. The enzymatic activity was eluted in a single peak starting after 150 ml of column eluate. 2.5.7. Second ammonium sulfate fractionation Fractions with the highest activity after filtration were subjected to a second ammonium sulfate fractionation. Three-fractions, 0 – 35%, 35 – 40% and 40–45% saturation were collected. All contained XDH activity but only 0 – 35 and 35 – 40% fractions were pure by electrophoresis. The enzyme resulting from DEAE-unbound fraction was not electrophoretically pure. In this case, a further preparative electrophoresis purification step was attempted but without positive results. 2.6. Analytical determinations Native molecular mass was determined by PAGE according to Hedrick and Smith [22] following the procedure indicated in Sigma Technical Bulletin No. MKR-137. Protein determination was performed according to Layne [23] and in pure preparations by the Murphy and Kies [24] spectrophotometric method using dialysis solution as blank. Spectrophotometric determinations were carried out in a Hitachi U-3200 spectrophotome-

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ter and fluorimetric analyses in a Jasco FP-750 spectrofluorometer at 20°C. Densitometric determinations were performed in a LKB 2202 Ultroscan Laser densitometer.

3. Results and discussion Xanthine oxidoreductase activity was very low in wheat leaves, about 1 kg of leaf material was required to obtain a total activity in the crude extract of 5.6 nkat with NAD + or 11.4 nkat with PMS as oxidizing substrates (Table 1). A purification procedure consisting of conventional fractionation and chromatographic steps (Table 1) resulted in xanthine dehydrogenase purification to electrophoretic homogeneity. As little as 0.5 mg of pure protein with 1–3 nkat activity was recovered from 1 kg wheat leaves at the end of purification. Pure enzyme specific activity expressed as uric acid produced was 6.5 and 1.8 nkat mg − 1 with PMS and NAD + , respectively (Table 1). With NAD + as acceptor the specific activity was 3.9 nkat mg − 1 if the initial rate (measured at 4 min of reaction) of NAD + reduction was considered. This value is comparable to that reported for Neurospora crassa XDH (2.53 nkat mg − 1) [25] and lower than those for soybean [14], bean root nodules [15], Chlamydomonas reinhardtii [10] and other XDHs of animal origin [17]. This low specific activity may be due to the intrinsic nature of the enzyme molecule or the presence of different inactive forms of the enzyme (demolybdo, desulpho), a well known aspect of xanthine oxidizing enzymes, particularly milk xanthine oxidase [26,27]. The extent of purification varied in relation to enzyme behaviour during anion exchange chromatography and only DEAE-cellulose bound fraction was purified 5000–9000-fold with a yield of about 25% (Table 1). After polyacrylamide gel electrophoresis under non-denaturing conditions, the native enzyme appeared homogeneous in only one protein band (Fig. 2B) while the enzyme fraction not retained by DEAE cellulose was only partially pure since there were three very close protein bands after electrophoresis and only one exhibited enzymatic activity (Fig. 2C). The ratio

5.60 5.00 3.20 2.60 1.66 1.54 1.50 0.90

11.40 11.00 12.19 10.59 10.40 8.40 6.14 3.34 18.86 125.2 635.59 1764.7

0.36 1.41 10.49

118.18 682.9 2601.7 6549.0

0.72 3.09 39.97

PMS

NAD+

NAD+ PMS

Specific activity (nkat mg−1)×1000

Total activity (nkat)b

52.4 347.8 1765.5 4901.9

1 3.9 29.1

NAD+

164.1 948.5 3613.5 9095.8

1 4.3 55.5

PMS

Purification factor

100 89 57 46 30 28 27 16

NAD+

100 96 107 93 91 74 54 29

PMS

Recovery (%)

b

This purification table refers to two preparations giving about 100% of the activity bound to DEAE cellulose. Data refer to 1 kg of fresh leaf tissue. Activity expressed as uric acid produced from xanthine in the standard reaction mixture with nicotinamide adenine dinucleotide (NAD+) and phenazine methosulfate (PMS) as acceptors. The reaction time was 3 h. Due to low activity in crude extract, in this case the time of reaction was 24 h.

a

15 752 3555 305

Crude extract Heat treatment Acetone fractionation Protamine precipitation DEAE-cellulose chromatography First ammonium sulfate fractionation Gel filtration Second ammonium sulfate fractionation 88 12.3 2.36 0.51

Total protein (mg)

Steps

Table 1 Purification of wheat leaf xanthine dehydrogenasea

94 P. Montalbini / Plant Science 134 (1998) 89–102

P. Montalbini / Plant Science 134 (1998) 89–102

Fig. 2. Visualization on polyacrylamide gel rods of purified wheat leaf xanthine dehydrogenase. Pure enzyme was subjected to electrophoresis under non-denaturing conditions in 7% polyacrylamide using a continuous system where the same buffer consisting of 50 mM Tris, 50 mM boric acid and 2.5 mM EDTA at pH 8.5 was used in the resolving gel and in the upper and lower electrode chambers. Stained for activity (A) as described in Section 2 or for protein with Commassie brilliant blue (B). Staining of protein after purification of enzyme not bound to DEAE cellulose is also reported (C).

between uric acid production with PMS and NAD + as acceptors was inconstant during purification. It was two in crude extracts and about four at the end of purification. Maybe the PMScatalyzed uric acid production by crude extract was underestimated since this acceptor is sensitive to components in crude extracts with consequent interference from the enzyme reaction. Enzyme activity was optimal between pH 8.0 and 8.6 with NAD + and PMS as oxidizing substrates. NADH diaphorase showed a first optimum in the same range followed by a second at 9.8 (Fig. 3). Native molecular mass determined by an electrophoretic method [22] was 350910 kDa.

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Purified and partially purified enzyme (Fig. 2) stored in TBEMM buffer in ice was stable for more than one year without appreciable loss of activity. The visible absorption spectrum of pure enzyme with a maximum at 329–332 nm, a shoulder at about 392–394 nm and low absorption peak from 480 to 510 nm (Fig. 4a) differs from those reported for other xanthine dehydrogenases and hydroxylases containing molybdenum, flavin and iron-sulphur clusters as prosthetic groups, usually characterized by peaks close to 450 nm with a shoulder at 550 nm [1,5,10]. A different protein structure and/or prosthetic group composition, demonstrated in Neurospora XDH [25], may be responsible for the atypical spectrum like those of xanthine oxidase from bovine small intestine [28] and heme-containing XDH from Pseudomonas putida [29]. Maximum absorption at about 320– 330 nm for the wheat leaf XDH may be due to a high level of pterins which have the same absorption maximum [30]. The fluorescence spectra of pure enzyme and partially purified preparation with a maximum emission at 484–486 nm (Fig. 4b) is similar to flavins and some flavoprotein

Fig. 3. Optimum pH of uric acid production from xanthine by wheat XDH with NAD + ( ) and PMS () as oxidizing substrates. NADH diaphorase activity ( š) measured by NBT reduction is also shown.

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Fig. 4. Absorption spectrum (a) and fluorescence emission spectrum (b) of wheat leaf xanthine dehydrogenase. Four emission peaks corresponding to excitation wavelengths of 380, 400, 405 and 412 nm are reported all showing maxima at 484 – 486 nm and increasing intensity (lowest with 420 and highest with 380 nm excitation wavelengths). A smaller emission with the same maximum was also obtained with an excitation wavelength of 310 nm (not shown). Both highly purified and partially purified enzyme (Fig. 2B, C) gave exactly the same spectral characteristics.

emission spectra, characterized by a broad band around 500 nm [31], indicating the presence of flavin in wheat leaf XDH molecule. Wheat leaf xanthine dehydrogenase catalyzed the transfer of electrons from xanthine to a variety of artificial acceptors (Table 2). Molecular oxygen alone did not accomplish substrate hy-

droxylation indicating that the wheat leaf xanthine oxidizing enzyme is a xanthine dehydrogenase strictu sensu similar to the Chlamydomonas XDH [9] and quite different from pea leaf XDH which have about 20% of oxidase activity [11]. The total ineffectiveness of NADP + , FAD and ferricyanide as acceptors also differenti-

P. Montalbini / Plant Science 134 (1998) 89–102

ates wheat leaf XDH from pea leaf and other xanthine oxidizing enzymes isolated from a wide array of animal species [32]. However, the high rate of xanthine oxidation in the presence of PMS, methylene blue and methyl viologen strongly differentiates wheat from the Chlamydomonas [9,10] enzyme. All these differences, particularly the very high rate of reaction accomplished with PMS, are presumably related to the nature and composition of the prosthetic groups of the enzyme and how reduction of acceptors is carried out by different prosthetic groups [10]. Wheat leaf XDH specifically catalyses the hydroxylation of xanthine and hypoxanthine and to a much lesser extent other purines, pteridines and aldehydes. It also catalyses the oxidation of NADH and NADPH (Table 3). Using a wide array of acceptors and a wide range of substrate concentrations, hypoxanthine exhibited a lower rate of oxidation compared to xanthine. Similar behaviour was reported for lentil [33] and Clostridium acidi urici [34] XDHs, but in general exactly the opposite is reported for all other XDHs [1,10,15]. Table 2 Electron acceptor utilization by wheat leaf XDH Electron acceptors

Concentration (mM)

Relative ratea

NAD NADP Phenazine mathosulfate (PMS) Methylene blue Nitrobluetetrazolium (NBT) Methyl viologen 2,6-Dichlorophenolindofenol (DCPIP) Ferricyanide Ferredoxin Flavin adenine dinucleotide (FAD) Oxygen

1.2 1.2 0.1

100 0 525

0.04 1.0

360 101

2.0 0.05

59 30

2.0 0.065 0.5

0 0 0

0.28

0

a

Activity measured as uric acid production with xanthine as substrate after 3-h reaction time. Data are the mean of three independent experiments performed with different enzyme preparations which, however, showed very similar properties with respect to electron acceptors.

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Table 3 Substrate specificity of wheat leaf XDH Substrate

Xanthine Hypoxanthine NADH NADPH Caffeine Theobromine Acetaldehyde Benzaldehyde Purine Pterine Allopurinol Oxypurinol Folic acid

XDH activity (relative rate) NBT reduction

NAD reduction

100 70 187 29 0 0 5 5 5 2 3 0.3 9

100 70

0 0 4 0 3.5 11 0 0 0

Acceptors reduction was determined spectrophotometrically at 540 (NBT) and 340 (NAD) nm.

The affinity of wheat leaf enzyme for reducing substrates (Table 4) was very high and the apparent Km values of 4–8 mM are in agreement with XDH from bean nodules [13] and Neurospora [25] and very close to those reported for other enzymes of plant and animal origin [17] but not lentil XDH [33]. Oxidizing substrate affinity of wheat leaf XDH (Table 4) showed a rather high Km for NAD + (120 mM) compared to those of similar enzymes from plant and animal sources [17] while the high affinity observed with PMS (5.6 mM) and NBT (11.3 mM) cannot be compared with other similar enzymes because there is no data available. Table 4 Apparent Km of wheat leaf xanthine dehydrogenase Substratea

Km (mM)b

Xanthine (NADH) Hypoxanthine (NADH) Xanthine (NBT reduced) Hypoxanthine (NBT reduced) NAD (NADH) PMS (uric acid) NBT (NBT reduced) NADH (NBT reduced)

8.2 4.1 8.6 7.5 171 5.6 11.3 5.7

a b

In parenthesis, the product of reaction measured is indicated. Obtained by plotting [s]/6 against [s] (Hanes-Woolf plot).

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P. Montalbini / Plant Science 134 (1998) 89–102

Fig. 5. Time course of uric acid production from xanthine with NAD + () and PMS ( ) as oxidizing substrates and time course of NAD + (š ) reduction with xanthine as reducing substrates. The effect of SOD and catalase (indicated in parenthesis) on PMS-catalyzed uric acid production from xanthine is also reported. The assay mixture (1.2 ml) of 0.13 M sodium pyrophosphate buffer (pH 8.0) containing 1 mM xanthine or hypoxanthine, 1 mM NAD + or 0.1 mM phenazine methosulphate (PMS) and 1 mg of enzyme. SOD or catalase effects were performed with 300 units of each enzyme added to the reaction mixture. NAD + reduction followed spectrophotometrically at 340 nm for 4 min with 1.5 mg of enzyme.

Like Chlamydomonas enzyme [10], superoxide dismutase and catalase have little or no effect on the reaction rate of wheat leaf XDH in the presence of NAD + as oxidizing substrate. However, with PMS as oxidizing substrate the rate of wheat leaf XDH activity was strongly enhanced 4 h after starting the reaction (Fig. 5). Therefore it can be presumed that the generation of oxygen and oxygen radicals in PMS-catalysed xanthine oxidation are responsible for inhibition and/or inactivation of the enzyme. The effects exerted by different inhibitors (Table 5), particularly p-hydroxymercuribenzoate (inhibitor of SH-groups) and salicylhydroxamic

acid (complexing reagent for non-heme iron) are typical for xanthine oxidases and dehydrogenases. The low inhibition by oxypurinol of PMS-catalyzed xanthine oxidation is not clearly understood but agrees with previous results obtained with xanthine oxidizing activity from various leaf materials [35,36]. Uric acid added to the reaction mixture at 1–2 mM concentration had a contrasting effect. NAD + reduction with xanthine was inhibited by 15% and with hypoxanthine enhanced by 30%. The effect of xanthine concentrations on the rate of XDH activity was determined by uric acid production with NAD + and PMS as oxidizing substrates. Both processes were strongly inhibited by high xanthine concentration, particularly with NAD + as acceptor. Optimal xanthine concentration was near 0.5 mM. A similar effect was observed measuring the initial rate of NAD + reduction (Fig. 6). Chlamydomonas XDH [10] is irreversibly inactivated by incubating the enzyme with its physiological electron donors xanthine and hypoxanthine. Preincubation of wheat leaf XDH with xanthine strongly inhibits uric acid production produced in 5-h reaction time (Table 6). At 20°C XDH activity was 70% inhibited by only 5-min preincubation with xanthine and almost completely inhibited (92%) with 30 min using NAD + as oxidizing substrate. With PMS as acceptor the inhibition was lower and the maximum effect was reached after 4 h. Substrate inhibition was strongly reduced and/or delayed if preincubation was performed at ice temperature (Table 6). In subsequent experiments the time course of NAD + reduction in 24-h reaction time was followed by preincubating the enzyme with xanthine and hypoxanthine. A high level of inhibition (Fig. 7) was maintained only during the first hours of reaction and full activity was autonomously restored after prolonged reaction time (between 10 and 24 h). Therefore, preincubation with reducing substrates is not an irreversible phenomenon. Since the enzyme activity was not completely inhibited even with a preincubation of 24 h (Table 6; Fig. 7), it seems likely that a certain reaction activity takes place under these inhibitory conditions and may give rise to reaction products in

P. Montalbini / Plant Science 134 (1998) 89–102

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Table 5 Effect of inhibitors on wheat leaf XDH Inhibitors

p-Hydroxymercuribenzoate

Salicylhydroxamic acid

o-Phenanthroline Diethyldithiocarbamate Sodium cyanide Allopurinol Oxypurinol Uric acid (xanthine) Uric acid (hypoxanthine)

Concentration (mM)

0.002 0.01 0.05 0.1 0.5 0.5 1 5 10 7.5 10 1.6 3.3 1 1 2 0.1–2

% Inhibition of XDH activity with the following acceptorsa NADb

PMSb

24 76 96 99 100 n.d. 3 29 52 88 28 n.d. 31 100 100 85 112 – 132

0 0 16 36 82 27 38 65 76 7 0 6 19 97 67 n.d. n.d.

a

Activity measured as uric acid production and expressed as percent of the corresponding controls without inhibitors. Only with uric acid the activity was determined by NAD+ reduction. b Data are the mean of five independent experiments performed with different enzyme preparations exibiting very similar behaviour. n.d., Not determined.

turn responsible for the enzyme autonomous reactivation. XDH activity measured as NAD + reduction under anaerobic conditions was about 30% the corresponding activity in air during the first 4 h of reaction. However, as the reaction proceeded the activity under anaerobic conditions increased, about 60% after 24 h (90% from 10 – 24 h) (Fig. 7). The low rate of NAD + reduction under anaerobiosis expressed by wheat leaf enzyme contrasts with pea leaf and Chlamydomonas XDHs. In conclusion, this general outline shows that xanthine oxidoreductase isolated from wheat leaves has common characteristics with similar enzymes isolated from different sources. However, unique features were also found, mainly in relation to visible spectrum and behaviour under anaerobiosis. Though inactivation of the enzyme by incubation with the substrate confirms previous results obtained with the algal enzyme, our

results indicate that this kind of inhibition and that obtained under anaerobiosis is restored autonomously beginning some hours after the reaction started. Similarly, the stimulatory effect exerted by SOD and catalase on PMS-catalysed uric acid production started after about 4-h reaction time. Therefore, it seems likely that certain intermediate reaction products during xanthine oxidation and acceptor reduction (probably oxygen and oxygen radical among them) may play a role when the appropriate concentration is reached in the reaction mixture. However, the inactivation mechanism of xanthine oxidizing enzymes is still not understood and many factors seem involved [10]. Further experiments should be performed to clarify these and other aspects. First of all, the exact nature of the prosthetic groups of wheat leaf XDH should be thoroughly verified, but to do this more amount of pure enzyme is required.

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Table 6 Percent XDH inhibition by incubation with the substrate (xanthine) before the reaction Acceptor

Incubation temperature (°C)

Incubation timea 5 min

PMS NAD

20 0 20 0

71

15 min

87

30 min

1h

4h

12 h

24 h

11 0 92 64

35 8 96 70

82 18 96 96

82 37 96 96

82 58 96 96

a

After enzyme incubation in buffer with and without xanthine, the reaction was started by acceptor. Reaction was stopped after 4 h and uric acid produced determined.

Acknowledgements This work was supported by a grant from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica Progetto nazionale: ‘Biodiversita` dei microrganismi fitopatogeni: variabilita` e interazioni con gli ospiti’ (ex M.U.R.S.T. 40%) e progetto di ricerca di ateneo (ex M.U.R.S.T. 60%). The author wishes to thank Professor M.

Fig. 6. Uric acid production as a function of xanthine concentration with PMS ( ) and NAD + () as oxidizing substrates. Reaction conditions as in Fig. 4; uric acid determined after 3-h reaction. Data expressed as percentage of the highest value in both reaction conditions. The same effect reported also with the initial rate (4 min) of NAD + reduction ( š ).

Pineda (Biochemistry Dept. Faculty of Science, Cordoba, Spain) for his suggestions regarding the manuscript.

Fig. 7. Wheat leaf XDH inactivation by xanthine. The enzyme (2 mg) was preincubated in the standard reaction mixture (1.2 ml) without NAD + for 5 min ( ), 1 h () and 16 h (). The reaction was started by adding NAD + (1 mM final concentration) and NAD + reduction estimated for 24 h at 340 nm. Data are expressed as percentage of activity observed with the enzyme not incubated with xanthine before starting the reaction. The numbers placed in the symbols indicate the percentage of activity calculated considering the interval with the previous determination (the number above the symbol at 24 h is the percentage of activity calculated from 10 to 24 h of reaction). The preincubation with hypoxanthine gave more marked effects (not shown). The effect of anaerobiosis (under helium atmosphere) on wheat XDH-catalysed xanthine oxidation, expressed as percent of NAD + reduction of control under air, is also reported (dotted line).

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