Functional expression of geraniol 10-hydroxylase reveals its dual function in the biosynthesis of terpenoid and phenylpropanoid

Geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase, has been reported to be involved in the biosynthesis of terpenoid indole alkaloids. The gene for Catharanthus roseus G10H (CrG10H) was cloned and heterologously expressed in baculovirus-infected insect cells. A number of substrates were subjected to assay the enzyme activity of CrG10H. As reported in a previous study, CrG10H hydroxylated the monoterpenoid geraniol at the C-10 position to generate 10-hydroxygeraniol. Interestingly, CrG10H also catalyzed 3'-hydroxylation of naringenin to produce eriodictyol. Coexpression of an Arabidopsis NADPH P450 reductase substantially increased the ability of CrG10H to hydroxylate naringenin. The catalytic activity of CrG10H was approximately 10 times more efficient with geraniol than with naringenin, judged by the k(cat)/K(m) values. Thus, G10H also plays an important role in the biosynthetic pathway of flavonoids, in addition to its previously described role in the metabolism of terpenoids.     

Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.     

Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution

A leucine aminopeptidase was purified for the first time from marine fish red sea bream ( Pagrus major) skeletal muscle to homogeneity with 4850-fold and a yield of 7.4%. The purification procedure consisted of ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, and phenyl-Sepharose. The enzyme was approximately 96 kDa as estimated by SDS-PAGE and gel filtration and preferentially hydrolyzed substrate Leu-MCA. The enzymatic activity was optimal at 45 degrees C and pH 7.5. The K m and k cat values of the enzyme for Leu-MCA were 1.55 microM and 26.4 S (-1) at 37 degrees C, respectively. Activation energy ( E a) of the enzyme was 59.6 kJ M (-1). The enzyme was specifically inhibited by metal-chelating agents, and Zn (2+) and (or) Mn (2+) seemed to be its metal cofactor(s). In addition, bestatin strongly inhibited its activity, and K i was 1.44 microM. Using a highly specific polyclonal antibody, the location of enzyme was demonstrated intracellularly and distributed in different tissues.     

Ascorbate regeneration by the reduced form of 2-amino-3-carboxy-1, 4-naphthoquinone, a strong growth stimulator for bifidobacteria

Nonenzymatic reduction of dehydroascorbate into ascorbate by the reduced form (quinol form) of 2-amino-3-carboxy-1,4-naphthoquinone, a strong growth stimulator for bifidobacteria, has been found. The bimolecular reaction rate constant was evaluated as 9 M(-)(1) s(-)(1) at pH 7.0. This reaction has been successfully coupled with enzymatic regeneration of the naphthoquinol by NAD(P)H in cell-free extracts of Bifidobacterium longum 6001. The overall reaction is a regeneration of NAD(P)(+) by dehydroascorbate [or a regeneration of ascorbate by NAD(P)H], in which the naphthoquinone/quinol redox couple functions as an electron transfer mediator. Kinetic study of the reduction of dehydroascorbate with related quinol compounds suggested the significance of the amino substituent of the naphthoquinol. A mechanism of the electron transfer from the quinol to dehydroascorbate is proposed, where the first step of the reaction is a nucleophilic addition of the C(2)-amino substituent of the naphthoquinol to the C(2)-position of dehydroascorbate to form a Schiff base intermediate.     

Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli

A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45 degrees C, respectively, and the enzyme maintained high activity at pH 5.0 and 60 degrees C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (k(cat)/K(M)) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached approximately 71% at 20 degrees C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for alpha-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.     

Effect of the Coenzymes NAD(P)(H) in Straight‐Dough Breadmaking on Protein Properties and Loaf Volume

The nicotinamide adenine dinucleotide coenzymes [NAD(P)(H)] are strong redox agents naturally present in wheat flour, and are indispensable cofactors in many redox reactions. Hence, it is not inconceivable that they affect gluten cross‐linking during breadmaking. We investigated the effect of increasing concentrations of NAD(P)(H) on gluten cross‐linking, dough properties, and bread volume using two flours of different breadmaking quality. Separate addition of the four nicotinamide coenzymes did not significantly affect mixograph properties. While addition of NAD⁺ hardly affected bread volume, supplementation with NADP(H) and NADH significantly decreased loaf volumes of breads made using flour of high breadmaking quality. Wheat flour incubation with NAD(P)H under anaerobic conditions increased wheat flour thiol content, while NAD(P)⁺ increased the extractability in SDS‐containing medium of the protein of the strong breadmaking flour. Based on the results, it was hypothesized that at least three reactions, competing for NAD(P)(H), occur during breadmaking that determine the final effect on protein, dough, and loaf properties. Next to coenzyme hydrolysis, the experiments pointed to coenzyme oxidation and NAD(P)(H) dependent redox reactions affecting protein properties.     

Hydrolysis of isoflavone glycosides by a thermostable β-glucosidase from Pyrococcus furiosus

The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purified with a specific activity of 330 U/mg for genistin by His-trap chromatography. The specific activity of the purified enzyme followed the order genistin > daidzin > glycitin> malonyl glycitin > malonyl daidzin > malonyl genistin. The hydrolytic activity for genistin was highest at pH 6.0 and 95 °C with a half-life of 59 h, a K(m) of 0.5 mM, and a k(cat) of 6050 1/s. The enzyme completely hydrolyzed 1.0 mM genistin, daidzin, and glycitin within 100, 140, and 180 min, respectively. The soybean flour extract at 7.5% (w/v) contained 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin. Genistin, daidzin, and glycitin in the soybean flour extract were completely hydrolyzed after 60, 75, and 120 min, respectively. Of the reported β-glucosidases, P. furiosusβ-glucosidase exhibited the highest thermostability, k(cat), k(cat)/K(m), yield, and productivity for hydrolyzing genistin. These results suggest that this enzyme may be useful for the industrial hydrolysis of isoflavone glycosides.     

Deactivation of ferrylmyoglobin by vanillin as affected by vanillin binding to β-lactoglobulin

Vanillin was found to be efficient as a deactivator of ferrylmyoglobin with a second-order rate constant of k(2) = 57 ± 1 L mol(-1) s(-1) for reduction to metmyoglobin with ΔH(?) = 58.3 ± 0.3 kJ mol(-1) and ΔS(?) = -14 ± 1 J mol(-1) K(-1) in aqueous pH 7.4 solution at 25 °C. Binding to β-lactoglobulin (βLG) was found to affect the reactivity of vanillin at 25 °C only slightly to k(2) = 48 ± 2 L mol(-1) s(-1) (ΔH(?) = 68.4 ± 0.4 kJ mol(-1) and ΔS(?) = 17 ± 1 J mol(-1) K(-1)) for deactivation of ferrylmyoglobin. Binding of vanillin to βLG was found to have a binding stoichiometry vanillin/βLG > 10 with K(A) = 6 × 10(2) L mol(-1) and an apparent total ΔH° of approximately -38 kJ mol(-1) and ΔS° = -55.4 ± 4 J mol(-1) K(-1) at 25 °C and ΔC(p, obs) = -1.02 kJ mol(-1) K(-1) indicative of increasing ordering in the complex, as determined by isothermal titration microcalorimetry. From tryptophan fluorescence quenching for βLG by vanillin, approximately one vanillin was found to bind to each βLG far stronger with K(A) = 5 × 10(4) L mol(-1) and a ΔH° = -10.2 kJ mol(-1) and ΔS° = 55 J mol(-1) K(-1) at 25 °C. The kinetic entropy/enthalpy compensation effect seen for vanillin reactivity by binding to βLG is concluded to relate to the weakly bound vanillin oriented through hydrogen bonds on the βLG surface with the phenolic group pointing toward the solvent, in effect making both ΔH(?) and ΔS(?) more positive. The more strongly bound vanillin capable of tryptophan quenching in the βLG calyx seems less or nonreactive.     

Evaluation of the dietetic and therapeutic potential of a high molecular weight hydroxycinnamate-derived polymer from Symphytum asperum Lepech. Regarding its antioxidant, antilipoperoxidant, antiinflammatory, and cytotoxic properties

A water-soluble hydroxycinnamate-derived polymer (>1000 kDa) from Symphytum asperum Lepech. (Boraginaceae) strongly reduced the diphenylpicrylhydrazyl radical (IC(50) approximately 0.7 microg/mL) and inhibited the nonenzymatic lipid peroxidation of bovine brain extracts (IC(50) approximately 10 ng). This polymer exhibited only a low hydroxyl radical scavenging effect in the Fe(3+)-EDTA-H(2)O(2) deoxyribose system (IC(50) > 100 microg/mL) but strongly decreased superoxide anion generation in either the reaction of phenazine methosulfate with NADH and molecular oxygen (IC(50) approximately 13.4 microg/mL) or in rat PMA-activated leukocytes (IC(50) approximately 5 microg/mL). The ability to inhibit both degranulation of azurophil granules and superoxide generation in primed leukocytes indicates that the NADPH oxidase responsible for this later effect is inhibited, pointing to the Symphytum asperum polymer as a potent antiinflammatory and vasoprotective agent. At all concentrations tested (0-200 microg/mL), we observed no cytotoxicity on normal human fibroblasts and neither antiproliferative effects nor proliferation activation on neoplastic cells.     

Purification and characterization of a hexanol-degrading enzyme extracted from apple

An enzyme having activity toward n-hexanol was purified from apple, and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 column. The obtained enzyme had a yield of 16.00% with a specific activity of 18879.20 U/mg protein and overall purification of 142.77-fold. The enzyme showed activity to isoamylol, 1-propanol, n-hexanol, and isobutanol but not toward methanol and ethanol. With n-hexanol as a substrate, the optimum conditions were pH 4.0 and 30 °C for enzyme activity and pH 3.0-4.0 and temperatures below 40 °C for enzyme stability. The enzyme activity was increased significantly by adding l-cysteine and Fe(2+) at all tested concentrations and slightly by Zn(2+) at a high concentration but decreased by additions of EDTA, Ga(2+), K(+), Mg(2+), sodium dodecyl sulfate (SDS), sodium aluminum sulfate (SAS), dithiothreitol (DTT), and glutathione (GSH). The enzyme activities toward n-hexanol and n-hexanal were increased by NADH but decreased by NAD(+), in contrast to a decrease toward n-hexane by addition of both NAD(+) and NADH.     

Comparison of three thermostable β-glucosidases for application in the hydrolysis of soybean isoflavone glycosides

A novel thermostable β-glucosidase (Te-BglA) from Thermoanaerobacter ethanolicus JW200 was cloned, characterized and compared for its activity against isoflavone glycosides with two β-glucosidases (Tm-BglA, Tm-BglB) from Thermotoga maritima. Te-BglA exhibited maximum hydrolytic activity toward pNP-β-d-glucopyranoside (pNPG) at 80 °C and pH 7.0, was stable for a pH range of 4.6-7.8 and at 65 °C for 3 h, and had the lowest K(m) for the natural glycoside salicin and the highest relative substrate specificity (k(cat)/K(m))((salicin))/(k(cat)/K(m))((pNPG)) among the three enzymes. It converted isoflavone glycosides, including malonyl glycosides, in soybean flour to their aglycons more efficiently than Tm-BglA and Tm-BglB. After 3 h of incubation at 65 °C, Te-BglA produced complete hydrolysis of four isoflavone glycosides (namely, daidzin, genistin and their malonylated forms), exhibiting higher productivity of genistein and daidzein than the other two β-glucosidases. Our results suggest that Te-BglA is preferable to Tm-BglA and Tm-BglB, but all three enzymes have great potential applications in converting isoflavone glycosides into their aglycons.     

The role of potassium in alleviating detrimental effects of abiotic stresses in plants

Plants exposed to environmental stress factors, such as drought, chilling, high light intensity, heat, and nutrient limitations, suffer from oxidative damage catalyzed by reactive oxygen species (ROS), e.g., superoxide radical (O₂equation/tex2gif-sup-1.gif^–), hydrogen peroxide (H₂O₂) and hydroxyl radical (OHequation/tex2gif-sup-4.gif). Reactive O₂ species are known to be primarily responsible for impairment of cellular function and growth depression under stress conditions. In plants, ROS are predominantly produced during the photosynthetic electron transport and activation of membrane‐bound NAD(P)H oxidases. Increasing evidence suggests that improvement of potassium (K)‐nutritional status of plants can greatly lower the ROS production by reducing activity of NAD(P)H oxidases and maintaining photosynthetic electron transport. Potassium deficiency causes severe reduction in photosynthetic CO₂ fixation and impairment in partitioning and utilization of photosynthates. Such disturbances result in excess of photosynthetically produced electrons and thus stimulation of ROS production by intensified transfer of electrons to O₂. Recently, it was shown that there is an impressive increase in capacity of bean root cells to oxidize NADPH when exposed to K deficiency. An increase in NADPH oxidation was up to 8‐fold higher in plants with low K supply than in K‐sufficient plants. Accordingly, K deficiency also caused an increase in NADPH‐dependent O₂equation/tex2gif-sup-6.gif^– generation in root cells. The results indicate that increases in ROS production during both photosynthetic electron transport and NADPH‐oxidizing enzyme reactions may be involved in membrane damage and chlorophyll degradation in K‐deficient plants. In good agreement with this suggestion, increases in severity of K deficiency were associated with enhanced activity of enzymes involved in detoxification of H₂O₂ (ascorbate peroxidase) and utilization of H₂O₂ in oxidative processes (guaiacol peroxidase). Moreover, K‐deficient plants are highly light‐sensitive and very rapidly become chlorotic and necrotic when exposed to high light intensity. In view of the fact that ROS production by photosynthetic electron transport and NADPH oxidases is especially high when plants are exposed to environmental stress conditions, it seems reasonable to suggest that the improvement of K‐nutritional status of plants might be of great importance for the survival of crop plants under environmental stress conditions, such as drought, chilling, and high light intensity. Several examples are presented here emphasizing the roles of K in alleviating adverse effects of different abiotic stress factors on crop production.     

Functional proteomic analysis of rice bran esterases/lipases and characterization of a novel recombinant esterase

An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 °C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 μM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) μM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.     

Comparison of the hydrolysis of bovine κ-casein by camel and bovine chymosin: a kinetic and specificity study

Bovine chymosin constitutes a traditional ingredient for enzymatic milk coagulation in cheese making, providing a strong clotting capacity and low general proteolytic activity. Recently, these properties were surpassed by camel chymosin, but the mechanistic difference behind their action is not yet clear. We used capillary electrophoresis and reversed-phase liquid chromatography-mass spectrometry to compare the first site of hydrolysis of camel and bovine chymosin on bovine κ-casein (CN) and to determine the kinetic parameters of this reaction (pH 6.5; 32 °C). The enzymes showed identical specificities, cleaving the Phe105-Met106 bond of κ-CN to produce para-κ-CN and caseinomacropeptide. Initial formation rates of both products validated Michaelis-Menten modeling of the kinetic properties of both enzymes. Camel chymosin bound κ-CN with ～30% lower affinity (K(M)) and exhibited a 60% higher turnover rate (k(cat)), resulting in ～15% higher catalytic efficiency (k(cat)/K(M)) as compared to bovine chymosin. A local, less dense negatively charged cluster on the surface of camel chymosin may weaken electrostatic binding to the His-Pro cluster of κ-CN to simultaneously impart reduced substrate affinity and accelerated enzyme-substrate dissociation as compared to bovine chymosin.     

Characterization of beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitor from Pueraria thunbergiana

This study describes the extraction and characterization of an inhibitor for beta-hydroxy-beta-methylglutaryl (HMG) coenzyme A (CoA) reductase from Pueraria thunbergiana. The maximum HMG-CoA reductase inhibitory activity (IC(50) = 79 microg) was obtained when P. thunbergiana was extracted with 70% ethanol at 30 degrees C for 12 h. After purification of the HMG-CoA reductase inhibitor by means of systematic solvent extraction, silica gel column chromatography, and HPLC, an active fraction with an IC(50) of 0.9 microg (4.25 microM) and a yield of 1.3% was obtained. The purified HMG-CoA reductase inhibitor was identified as daidzein (C(15)H(10)O(4); molecular mass, 254 Da).     

Aroma biosynthesis in strawberry: s-adenosylmethionine:furaneol o-methyltransferase activity in ripening fruits

Among the most important volatile compounds in the aroma of strawberries are 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) and its methoxy derivative (methoxyfuraneol, mesifuran). Three strawberry varieties, Malach, Tamar, and Yael, were assessed for total volatiles, Furaneol, and methoxyfuraneol. The content of these compounds sharply increased during fruit ripening, with maximum values at the ripe stage. An enzymatic activity that transfers a methyl group from S-adenosylmethionine (SAM) to Furaneol sharply increases during ripening of strawberry fruits. The in vitro generated methoxyfuraneol was identified by radio-TLC and GC-MS. The partially purified enzyme had a native molecular mass of approximately 80 kDa, with optimum activity at pH 8.5 and 37 degrees C. A high apparent K(m) of 5 mM was calculated for Furaneol, whereas this enzyme preparation apparently accepted as substrates other o-dihydroxyphenol derivatives (such as catechol, caffeic acid, and protocatechuic aldehyde) with much higher affinities (K(m) approximately 105, 130, and 20 microM, respectively). A K(m) for SAM was found to be approximately 5 microM, regardless of the acceptor used. Substrates that contained a phenolic group with only one OH group, such as p-coumaric and trans-ferulic acid, as well as trans-anol and coniferyl alcohol, were apparently not accepted by this activity. It is suggested that Furaneol methylation is mediated by an O-methyltransferase activity and that this activity increases during fruit ripening.     

Action of tyrosinase on ortho-substituted phenols: possible influence on browning and melanogenesis

The action of tyrosinase on ortho-substituted monophenols (thymol, carvacrol, guaiacol, butylated hydroxyanisole, eugenol, and isoeugenol) was studied. These monophenols inhibit melanogenesis because they act as alternative substrates to L-tyrosine and L-Dopa in the monophenolase and diphenolase activities, respectively, despite the steric hindrance on the part of the substituent in ortho position with respect to the hydroxyl group. We kinetically characterize the action of tyrosinase on these substrates and assess its possible effect on browning and melanognesis. In general, these compounds are poor substrates of the enzyme, with high Michaelis constant values, K(m), and low catalytic constant values, k(cat), so that the catalytic efficiency k(cat)/K(m) is low: thymol, 161 ± 4 M(-1) s(-1); carvacrol, 95 ± 7 M(-1) s(-1); guaiacol, 1160 ± 101 M(-1) s(-1).     

Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. amiga)

Three phytases were purified about 14200-fold (LP11), 16000-fold (LP12), and 13100-fold (LP2) from germinated 4-day-old lupine seedlings to apparent homogeneity with recoveries of 13% (LP11), 8% (LP12), and 9% (LP2) referred to the phytase activity in the crude extract. They behave as monomeric proteins of a molecular mass of about 57 kDa (LP11 and LP12) and 64 kDa (LP2), respectively. The purified proteins belong to the acid phytases. They exhibit a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 80 microM (LP11), 300 microM (LP12), and 130 microM (LP2) and k(cat) = 523 s(-1) (LP11), 589 s(-1) (LP12), and 533 s(-1) (LP2) at pH 5.0 and 35 degrees C. The phytases from lupine seeds exhibit a broad affinity for various phosphorylated compounds and hydrolyze phytate in a stepwise manner.     

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda)

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.     

Influence of pH and soil copper on adsorption of metalaxyl and penconazole by the surface layer of vineyard soils

The upper horizons of old vineyard soils have substantial copper contents due to the traditional use of copper-based fungicides. Total copper levels in eight vineyard soils in the Rías Baixas area of Galicia (northwestern Spain) ranged from 60 to 560 mg kg(-1) (mean +/- SD = 206 +/- 170 mg kg(-1)). The adsorption of the fungicides metalaxyl (pK(a) = 1.41) and penconazole (pK(a) = 2.83) by these soils was determined using fungicide solutions of pH 2.5 and 5.5, and desorption of fungicide adsorbed at pH 5.5 was also determined. In all cases, Freundlich equations were fitted to the data with R (2) > 0.96. Penconazole was adsorbed and retained more strongly than metalaxyl, with K(F) values more than an order of magnitude greater. In the desorption experiments, both fungicides exhibited hysteresis. Soil copper content hardly affected the adsorption of metalaxyl, but K(F) values for adsorption of penconazole increased at a rate of about 0.1 mL(n) (microg of penconazole)(1-n) (microg of Cu)(-1), which is attributed to the formation of Cu(2+)-penconazole complexes with greater affinity for soil colloids than penconazole itself. Because the dependence of K(F) for penconazole adsorption on copper content was the same at both pH values, complex formation appears not to have been affected by the solubilization of 6-17% of soil copper at pH 2.5. A similar copper dependence, or lack of dependence, was observed when 100-1000 mg kg(-1) of copper was added as Cu(NO(3))(2).2H(2)O to the solutions from which the fungicides were adsorbed.