High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other ribonucleoside diphosphates with nanomolar sensitivity

A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.     

Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase.

Mycobacterium tuberculosis ribonucleotide reductase (RNR) is a potential target for new antitubercular drugs. Herein we describe the synthesis and evaluation of peptide inhibitors of RNR derived from the C-terminus of the small subunit of M. tuberculosis RNR. An N-terminal truncation, an alanine scan and a novel statistical molecular design (SMD) approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied in this study. The alanine scan showed that Trp5 and Phe7 were important for inhibitory potency. A quantitative structure relationship (QSAR) model was developed based on the synthesized peptides which showed that a negative charge in positions 2, 3, and 6 is beneficial for inhibitory potency. Finally, in position 5 the model coefficients indicate that there is room for a larger side chain, as compared to Trp5.     

Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

Removal of beta-glucanase activities from commercial amyloglucosidase by chromatography on cellulose powder

The amount of CO bound to hemocyanin has been determined by titration with human hemoglobin (Hb). The spectrophotometric method makes use of the high affinity of Hb for CO, which (at pH 9) allows a complete transfer of the ligand from hemocyanin to hemoglobin. Taking advantage of the large spectral changes at the level of the heme, this method allows the rapid determination of the CO bound to hemocyanin, using relatively small amounts of protein (less than 5 mg). Application of this method shows (i) that CO binds to hemocyanins with a stoichiometry of 1:1, in agreement with one earlier observation with ¹⁴C-labeled carbon monoxide (1), and (ii) that decrease of the CO-copper luminescence of hemocyanin observed upon addition of KCN is quantitatively related to the displacement of CO from the active site.     

Iron limitation results in induction of ferricyanide reductase and ferric chelate reductase activities in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii Dangeard CW-15 exhibited very low rates of plasma-membrane Fe(III) reductase activity when grown under Fe-sufficient conditions. After switching the medium to an Fe-free formulation, both ferricyanide reductase and ferric chelate reductase activities rapidly increased, reaching a maximum after 3 d under iron-free conditions. Both of the Fe(III) reductase activities increased in parallel over time, they exhibited similar K _m values (approximately 10 μM) with respect to Fe(III), displayed the same pH profile of activity, and both exhibited the same degree of light stimulation which could be inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). Furthermore, ferricyanide competitively inhibited ferric chelate reduction by iron-limited cells. These results indicate that both Fe(III) reductase activities were mediated by the same iron-limitation-induced plasma-membrane reductase. No evidence was found for the presence of Fe(III)-reducing substances in the culture medium, or for the involvement of active oxygen species in the process of Fe(III) reduction. Chlamydomonas reinhardtii appears to respond to iron limitation in a manner similar to Strategy I higher plants. Received: 24 June 1997 / Accepted: 2 August 1997     

Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4′-sulfonyl derivative of l-methionine (dabsyl Met), the product of the enzymatic reactions when either dabsyl l-methionine S-sulfoxide or dabsyl l-methionine R-sulfoxide is used as a substrate. The method provides baseline resolution of the substrates and, therefore, can be used to easily determine the purity of the substrates. The method is rapid (∼20 min sample to sample), requires no column regeneration, and uses very small amounts of buffers. Separation was performed by using a 75-μm internal diameter polyimide-coated fused silica capillary (no inside coating) with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV, and the separation buffer was 25 mM KH₂PO₄ (pH 8.0) containing 0.9 ml of N-lauroylsarcosine (sodium salt, 30% [w/v] solution) per 100 ml of buffer. Prior to use, the capillary was conditioned with the same buffer that also contained 25 mM sodium dodecyl sulfate. The CE method is compared with high-performance liquid chromatography (HPLC) as determined by comparing results from measurements of hepatic enzyme activities in mice fed either deficient or adequate selenium.     

Induction of nitrate reductase, nitrite reductase, and glutamine synthetase isoforms in sunflower cotyledons as affected by nitrate, light, and plastid integrity

Summary We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO ₃ ^– , NO ₂ ^– or NH ₄ ⁺ ), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO ₂ ^– or NH ₄ ⁺ the synthesis of NiR and chloroplastic GS (GS₂) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO ₃ ^– concentration indicate that NO ₃ ^– -dependent enzyme induction is elicited by NO ₃ ^– per se and not by a product of its assimilatory reduction, e.g., NO ₂ ^– or NH ₄ ⁺ . In the presence of NO ₃ ^– , light remarkably enhanced the appearance of NR, NiR, and GS₂, while the activity of the cytosolic GS isoform (GS₁) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light- and NO ₃ ^– -dependent increase of GS₂ activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO ₃ ^– on the appearance of NR, NiR, and GS₂ isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO ₃ ^– and/or for the accumulation of newly formed enzymes in the organelle.Abbreviations CAP chloramphenicol - CHX cycloheximide - GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic (chloroplastic) GS - NF norflurazon - NiR nitrite reductase - NR nitrate reductase     

Fluoride,beryllium and ADP combine as a ternary complex in aqueous solution as revealed by a multinuclear NMR study

Different kinds of nucleotide binding enzymes are sensitive to fluoroberyllate complexes (BeF.) and fluoroaluminate complexes (AlF_y). It has been hypothesized that the effects of these fluorometals are related to the generation at a nucleotide binding site of a pseudo nucleoside triphosphate, consisting of a fluorometal moiety bound to the phosphate group of a molecule of nucleoside diphosphate (Bigay et al. 1985; Lunardi et al. 1985). In order to establish whether ternary complexes comprising ADP, beryllium and fluoride can exist in slightly alkaline solution in the absence of enzyme, we have carried out a multinuclear (³¹P, ⁹Be and ^t9F) NMR study. In preliminary experiments, pyrophosphate (PPi) was substituted for ADP and taken as a simpler analog of nucleoside diphosphate. In the absence of fluoride, three types of PPi-Be complexes were generated: two of these were bidentate chelates with either one or two pyrophosphate molecules bound per beryllium; the third one was a monodentate complex. It is probable that the same types of combination exist between the polyphosphate chain of ADP and Be. In the presence of fluoride, both ADP and PPi combined with beryllium to form ternary complexes. These complexes consisted of monofluoroberyllate(-BeF) or difluoroberyllate (-BeF,) bound to the two phosphates of one molecule of ADP or PPi as a bidentate chelate. We failed to observe the formation of complexes between ADP and trifluoroberyllate (-BeF₃). The relevance of this study to the biological effects of fluoride and beryllium on various enzymic reactions is discussed.Abbreviations PPi pyrophosphate - AMP adenosine -5-monophos-phate - ADP adenosine- 5-diphosphate - ADPS adenosine-5-O-(2-thiodiphosphate) - Ap²A P1,p²-di(adenosine-5)pyrophosphate - F₁-ATPase catalytic sector (soluble) of the beef heart mitochondrial ATPase complex - Tris tris(hydroxymethyl)aminomethane Offprint requests to: J.-L. Girardet     

Effect of salicylic acid on nitrite reductase and glutamate dehydrogenase activities in maize roots

The effects of 0.01 to 5 m M salicyclic acid on the increase in nitrite reductase or glutamate dehydrogenase activities in maize roots by nitrate or ammonium respectively, were examined. Nitrite reductase activity was inhibited by the highest concentration of the acid. The activity of NADH-glutamate dehydrogenase was stimulated slightly (but consistently) by the lowest concentration and was inhibited by higher concentrations. Total protein content was also inhibited at high concentrations. When the crude enzyme extract was stored at 25°C in light, the glutamate dehydrogenase activity in the control decreased after 4 h of incubation. Low concentrations of the acid had no effect on this decrease but higher concentration accelerated the process. The divalent cations Caz²⁺, Mn²⁺, Mg²⁺ and Zn²⁺ protected against loss of enzyme activity during storage, both in the absence and presence of the acid. The inhibitory effect of 5 m M salicylic acid on glutamate dehydrogenase activity is apparent due to interference with the activity of the enzyme rather than with its synthesis.     

Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography.

A scheme is described for the large scale purification of thioredoxin, thioredoxin reductase, and glutathione reductase. The scheme is based on an initial separation of thioredoxin from the two reductases by affinity chromatography on agarose-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (agarose-2',5'-ADP). The two reductases were then separated by hydrophobic chromatography and purified separately to homogeneity. Thioredoxin was purified to homogeneity by immunoadsorption to agarose containing immobilized goat anti-thioredoxin. Overall yields for thioredoxin, thioredoxin reductase, and glutathione reductase exceeded 80% in each case. Both reductases exhibit an absorption band at approximately 320 nm which appears due to a residual amount of tightly bound NADP. Presence of this absorption band has no apparent effect on the specific activity of either enzyme.     

Determination of the distribution of three nitrate reductase isoforms in soybean seedlings by chromatography and a simple method based on assay conditions

Blue Sepharose affinity chromatography was used to study the distribution of the constitutive NAD(P)H-nitrate reductase (EC 1.6.6.2: Cl-NR) and of the constitutive and inducible NADH-nitrate reductases (EC 1.6.6.1; C2-NR and i-NR, respectively), in the unifoliolate leaf (F0), the first and the second trifoliolate leaves (F1 and F2) and the roots of urea- and nitrate-grown soybean ( Glycine max [L.] Merr.) plants. The C1-NR eluted by NADPH is present in the F0 and F1 leaves and nearly absent in the F2 leaf. The activity pattern of this isoform is not modified by nitrate nutrition. The C2-NR eluted by NADH is high in the F0 leaf, low in the F1 leaf and nearly absent in the F2 leaf of urea-grown plants. The NADH elution from leaves of nitrate-grown plants is a mixture of C2-NR and i-NR, requiring careful interpretation of results. However, i-NR appears the principal isoform in the leaves especially in the F2 leaf. This i-NR is the only NR present in the roots.
The pH effect on the assay of the 3 partially purified isoforms was studied using LNR2 and LNR5 soybean mutants to remove the cross contamination. It appears that C1-NR and C2-NR activities are negligible at pH 8.5, which allows the assay of only the i-NR in a crude extract at this pH, even when C1-NR and C2-NR are present. It appears also that the assay of C1-NR activity at pH 6.5 with NADPH is free of interference by the i-NR. To estimate the C2-NR activity with NADH at pH 6.5 in a crude extract in the presence of C1-NR and i-NR, we propose a simple calculation using the coefficient from the pH responses. These calculations are used to compare the development of C1-NR, C2-NR and i-NR activities in the F0 and F1 leaves of plants previously grown on urea and transferred to nitrate. Only the activity of the inducible isoform is modified by the nitrogen treatment. Activity of the constitutive isofroms appear stable during the 48 h treatment, with only a slight decrease in C1-NR activity being observed with time.     

Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species

BACKGROUND AND AIMS: Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. METHODS: Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. RESULTS: Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. CONCLUSIONS: V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.     

Inhibition of chlamydial class Ic ribonucleotide reductase by C‐terminal peptides from protein R2

Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese–iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C‐terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical‐containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese–iron cofactor is similar to that of the tyrosyl‐radical‐containing RNRs, involving reversible long‐range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2‐derived oligopeptide blocks the transfer pathway. We have constructed three‐dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20‐mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C‐terminus of protein R2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH₄⁺ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO₃⁺ in the presence of 0, 1 or 10 mM NH₄⁺, Exogenous NH₄⁺ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO₃⁺, In contrast, the induction of NiRA was inhibited by NH₄⁺ at all NO₃⁺ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH₄⁺ Pre-treatment with NH₄⁺ had no effect on the subsequent induction of NRA in the absence of additional NH₄⁺ whereas the induction of NiRA in NH₄⁺-pretreated roots was inhibited in the absence of NH₄⁺ At 10 mM NO₃⁺ L-methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH₄⁺ was present. In contrast, the induction of NiRA was inhibited by L-methionine sulfoximine irrespective of NH₄⁺ supply. During the postinduction phase, exogenous NH₄⁺ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH₃⁺ whereas, NiRA was unaffected by NH₄⁺ at either substrate concentration. The results indicate that exogenous NH₄⁺ regulates the induction of NRA in roots by limiting the availability of NO₃⁺. Conversely, it has a direct effect, independent of the availability of NO₃⁺, on the induction of NiRA. The lack of an NH₄⁺ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.     

Inhibition of aldose reductase by dietary antioxidant curcumin: Mechanism of inhibition, specificity and significance

Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. In this study we show that curcumin inhibits ALR2 with an IC₅₀ of 10 μM in a non-competitive manner, but is a poor inhibitor of closely-related members of the aldo-keto reductase superfamily, particularly aldehyde reductase. Results from molecular docking studies are consistent with the pattern of inhibition of ALR2 by curcumin and its specificity. Moreover, curcumin is able to suppress sorbitol accumulation in human erythrocytes under high glucose conditions, demonstrating an in vivo potential of curcumin to prevent sorbitol accumulation. These results suggest that curcumin holds promise as an agent to prevent or treat diabetic complications.     

The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75?U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I₅₀ and K_i values were 12.179?mM and 6.5123±4.1139?mM for cefotaxime, and 1.682?mM and 0.7446±0.2216?mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300?mg/kg cefotaxime and 1000?mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2?h (p<0.01). Cefotaxime led to increased enzyme activity at 4?h (p<0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6?h (p>0.05).     

Effects of cadmium and lead on ferric chelate reductase activities in sugar beet roots

The effects of the heavy metals Cd and Pb on the activity of the enzyme ferric chelate reductase (FC-R, E.C. 1.6.99.13) have been studied in excised sugar beet root tips. The activity of this enzyme is markedly increased by iron deficiency. Metals were used as chloride salts or chelated with EDTA, and chemical speciation was carried out to predict the metal chemical species in equilibrium both in the ferric reductase assay and in the nutrient solutions. Three different heavy metal treatments were used. First, effects of Cd and Pb on the functioning of the FC-R were assessed in Fe-deficient plants, by including metals in the enzyme assay medium only. Results indicate that 50 μM CdCl₂ or Cd-EDTA did not affect FC-R activities even when assay time was as long as 2 h, whereas Pb slightly decreased enzyme activity only at concentrations of 2 mM. Second, short-time Cd and Pb pre-treatments (30–60 min) were imposed on intact Fe-deficient plants before carrying out the assay of FC-R activity. These short-term treatments induced significant decreases in the FC-R activities previously induced by Fe deficiency. With Cd, effects were more pronounced at higher concentrations, and they were stronger when Cd was in the free ion form than when present in the form of Cd-EDTA chelate. Third, prolonged Cd and Pb treatments were imposed on plants grown on 45 μM Fe-EDTA to assess the long-term effects of heavy metals on the induction of the FC-R enzyme. These long-term heavy metal treatments caused a significant increase in the root FC-R activities, indicating that Cd and Pb induce a deficiency in Fe in sugar beet that in turn elicits FC-R activity. The increases, however, are not as large as those found in total absence of Fe.