acid-phosphatase and arsenic-acid

Metal(loid)s can promote the spread and enrichment of antibiotic resistance in the environmental ecosystem through a co-selection effect. Little is known about the ecological effects of entering antibiotics into the environment with long-term metal(loid)s' resistance profiles. Here, cow manure containing oxytetracycline (OTC) or sulfadiazine (SA) at four concentrations (0 (as control), 1, 10, and 100 mg/kg) was loaded to a maize cropping system in an area with high a arsenicals geological background. Results showed that exogenous antibiotics entering significantly changed the nutrient conditions, such as the concentration of nitrate nitrogen, ammonium nitrogen, and available phosphorus in the maize rhizosphere soil, while total arsenic and metals did not display any differences in antibiotic treatments compared with control. Antibiotics exposure significantly influenced nitrate and nitrite reductase activities to reflect the inhibition of denitrification rates but did not affect the soil urease and acid phosphatase activities. OTC treatment also did not change soil dehydrogenase activities, while SA treatment posed promotion effects, showing a tendency to increase with exposure concentration. Both the tested antibiotics (OTC and SA) decreased the concentration of arsenite and arsenate in rhizosphere soil, but the inhibition effects of the former were higher than that of the latter. Moreover, antibiotic treatment impacted arsenite and arsenate levels in maize root tissue, with positive effects on arsenite and negative effects on arsenate. As a result, both OTC and SA treatments significantly increased bioconcentration factors and showed a tendency to first increase and then decrease with increasing concentration. In addition, the treatments decreased translocation capacity of arsenic from roots to shoots and showed a tendency to increase translocation factors with increasing concentration. Microbial communities with arsenic-resistance profiles may also be resistant to antibiotics entering.

Topics: Acid Phosphatase; Ammonium Compounds; Anti-Bacterial Agents; Arsenates; Arsenic; Arsenicals; Arsenites; Ecosystem; Manure; Nitrates; Nitrite Reductases; Nitrogen; Oxidoreductases; Oxytetracycline; Phosphorus; Rhizosphere; Soil; Sulfadiazine; Urease; Zea mays

The physiological responses of the arsenic-hyperaccumulator, Pteris vittata, such as arsenic uptake and chemical transformation in the fern, have been investigated. However, a few questions remain regarding arsenic treatment in hydroponics. Incubation conditions such as aeration, arsenic concentration, and incubation period might affect those responses of P. vittata in hydroponics. Arsenite uptake was low under anaerobic conditions, as previously reported. However, in an arsenite uptake experiment, phosphorous (P) starvation-dependent uptake of arsenate was observed under aerobic conditions. Time course-dependent analysis of arsenite oxidation showed that arsenite was gradually oxidized to arsenate during incubation. Arsenite oxidation was not observed in any of the control conditions, such as exposure to a nutrient solution or to culture medium only, or with the use of dried root; arsenite oxidation was only observed when live root was used. This result suggests that sufficient aeration allows the rhizosphere system to oxidize arsenite and enables the fern to efficiently take up arsenite as arsenate. X-ray absorption near edge structure (XANES) analyses showed that long-duration exposure to arsenic using a hydroponic system led to the accumulation of arsenate as the dominant species in the root tips, but not in the whole roots, partly because up-regulation of arsenate uptake by P starvation of the fern was caused and retained by long-time incubation. Analysis of concentration-dependent arsenate uptake by P. vittata showed that the uptake switched from a high-affinity transport system to a low-affinity system at high arsenate concentrations, which partially explains the increased arsenate abundance in the whole root.

Topics: Absorption; Acid Phosphatase; Aerobiosis; Anaerobiosis; Arsenates; Arsenites; Biological Transport; Hydroponics; Oxidation-Reduction; Phosphates; Phosphorus; Plant Roots; Pteris; Rhizosphere; Soil Pollutants

Cyclic voltammetry of the non-heme diiron enzyme porcine purple acid phosphatase (uteroferrin, Uf) has been reported for the first time. Totally reversible one-electron oxidation responses (FeIII-FeII --> FeII-FeIII) are seen both in the absence and in the presence of weak competitive inhibitors phosphate and arsenate, and dissociation constants of these oxoanion complexes formed with uteroferrin in its oxidized state (Uf(o)) have been determined. The effect of pH on the redox potentials has been investigated in the range 3 < pH < 6.5, enabling acid dissociation constants for Uf(o) and its phosphate and arsenate complexes to be calculated.

Topics: Acid Phosphatase; Animals; Arsenates; Binding, Competitive; Electrochemistry; Electron Transport; Enzyme Inhibitors; Glycoproteins; Hydrogen-Ion Concentration; Iron; Oxidation-Reduction; Phosphates; Swine

Despite the current availability of several crystal structures of purple acid phosphatases, to date there is no direct evidence for solvent-derived ligands occupying terminal positions in the active enzyme. This is of central importance, because catalysis has been shown to proceed through the direct attack on a metal-bound phosphate ester by a metal-activated solvent-derived moiety, which has been proposed to be either (i) a hydroxide ligand terminally bound to the ferric center or (ii) a bridging hydroxide. In this work we use (2)H Q-band (35 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy to identify solvent molecules coordinated to the active mixed-valence (Fe(3+)Fe(2+)) form of the dimetal center of uteroferrin (Uf), as well as to its complexes with the anions MoO(4), AsO(4), and PO(4). The solvent-derived coordination of the dinuclear center of Uf as deduced from ENDOR data includes a bridging hydroxide and a terminal water/hydroxide bound to Fe(2+) but no terminal water/hydroxide bound to Fe(3+). The terminal water is lost upon anion binding while the hydroxyl bridge remains. These results are not compatible with a hydrolysis mechanism involving a terminal Fe(3+)-bound nucleophile, but they are consistent with a mechanism that relies on the bridging hydroxide as the nucleophile.

Topics: Acid Phosphatase; Arsenates; Binding Sites; Catalysis; Hydrolases; Iron; Isoenzymes; Metalloproteins; Molybdenum; Nuclear Magnetic Resonance, Biomolecular; Phosphates; Solvents; Tartrate-Resistant Acid Phosphatase; Water

Bovine spleen purple acid phosphatase (BSPAP) is a dinuclear iron protein with two stable redox states. The Fe3+Fe2+ state is the active state, while the fully oxidized protein (BSPAPox) has been reported to retain 5-10% activity, corresponding to a kcat of ca. 150 s-1 [Dietrich, M., Münstermann, D., Suerbaum, H., and Witzel, H. (1991) Eur. J. Biochem. 199, 105-113]. Here we show that this activity does not originate from Fe3+Fe3+-BSPAP, but rather from an 'impurity' of FeZn-BSPAP. The FeZn form of BSPAP was prepared from apo-BSPAP following a new procedure, and its kinetic properties were carefully determined for comparison to those of BSPAPox. For the hydrolysis of p-NPP at pH 6.00, both kcat and KM were affected by the Fe2+-to-Zn2+-substitution [Fe3+Fe2+-BSPAP, kcat = (1.8 +/- 0.1) x 10(3) s-1 and KM = 1.2 +/- 0.2 mM; Fe3+Zn2+-BSPAP; kcat = (2.8 +/- 0.2) x 10(3) s-1 and KM = 3.3 +/- 0.4 mM]. The KM of BSPAPox was the same as that of FeZn-BSPAP. pH profiles of BSPAPox and FeZn-BSPAP were both shifted to lower pH compared to that of BSPAPred. FeZn-BSPAP, FeZn-BSPAP.PO4, and FeZn-BSPAP.MoO4 all showed characteristic EPR spectra similar to the corresponding complexes of FeZn-Uf. The same species could also be observed in concentrated samples of native BSPAP. Spin integration of these spectra showed a quantitative relation between the spin concentration of the FeZn-BSPAP 'impurity' and the residual phosphatase activity after oxidation. Since all activity found after oxidation of BSPAP could be attributed to FeZn-BSPAP, there is no direct evidence that Fe3+Fe3+-BSPAP is catalytically active. These results set an upper limit to the possible catalytic activity of the Fe3+Fe3+ form of

Topics: Acid Phosphatase; Animals; Apoenzymes; Arsenates; Binding, Competitive; Catalysis; Cattle; Edetic Acid; Electron Spin Resonance Spectroscopy; Enzyme Activation; Ferric Compounds; Glycoproteins; Kinetics; Molybdenum; Oxidation-Reduction; Phosphates; Spleen; Tungsten Compounds; Zinc

The PHO84 gene in Saccharomyces cerevisiae encodes a Pi transporter, mutation of which confers constitutive synthesis of repressible acid phosphatase (rAPase), in medium containing repressible amounts of Pi, and an arsenate-resistant phenotype. We selected an arsenate-resistant mutant showing the constitutive synthesis of rAPase on nutrient plates containing 4.5 mM arsenate. This mutant has double mutations designated as pho86 and pho87. The mutant transcribes PHO84 even in the repressible condition but has a severe defect in Pi uptake. The constitutive rAPase+ phenotype of the pho86 pho87 mutant was partially suppressed by an increased dosage of the PHO84 gene. The PHO87 gene was found to be identical with YCR524, according to the published nucleotide sequence of chromosome III, which encodes a protein of 923 amino-acid residues with a highly charged N-terminal half followed by a C-terminal half consisting of 12 membrane-spanning segments as in Pho84p. These and the other findings suggest that the Pho86p and Pho87p proteins collaborate with Pho84p in Pi uptake.

Topics: Acid Phosphatase; Amino Acid Sequence; Arsenates; Carrier Proteins; Drug Resistance; Fungal Proteins; Genes, Fungal; Membrane Proteins; Molecular Sequence Data; Mutation; Phosphate-Binding Proteins; Saccharomyces cerevisiae

Uteroferrin, the purple acid phosphatase from porcine uterine fluid, is noncompetitively inhibited by vanadate in a time-dependent manner under both aerobic and anaerobic conditions. This time-dependent inhibition is observed only with the diiron enzyme and is absent when the FeZn enzyme is used. The observations are attributed to the sequential formation of two uteroferrin-vanadium complexes. The first complex forms rapidly and reversibly, while the second complex forms slowly and results in the production of catalytically inactive oxidized uteroferrin and V(IV), which is observed by EPR. The redox reaction can be reversed by treatment of the oxidized enzyme first with (V(IV)) and then EDTA to generate a catalytically active uteroferrin. Multiple inhibition kinetics suggests that vanadate is mutually exclusive with molybdate, tungstate, and vanadyl cation. The binding site for each of these anions is distinct from the site to which the competitive inhibitors phosphate and arsenate bind. The time-dependent inhibition by vanadate of uteroferrin containing the diiron core represents a new type of mechanism by which vanadium can interact with proteins and gives additional insight into the binding of anions to uteroferrin.

Topics: Acid Phosphatase; Animals; Anions; Arsenates; Binding Sites; Cations; Edetic Acid; Electron Spin Resonance Spectroscopy; Female; Iron; Isoenzymes; Kinetics; Metalloproteins; Molybdenum; Oxidation-Reduction; Phosphates; Rhenium; Swine; Tartrate-Resistant Acid Phosphatase; Tungsten; Tungsten Compounds; Vanadates; Vanadium

The theory of multiple inhibition kinetics has been extended to enzymes for which one inhibitor is noncompetitive and the other exhibits mixed inhibition. Plots of reciprocal velocity versus the concentration of either inhibitor at various fixed concentrations of the second inhibitor are predicted to give parallel lines if binding of the inhibitors is mutually exclusive and intersecting lines if the inhibitors interact at different sites on the enzyme. Application of this analysis to the purple acid phosphatase from bovine spleen in the presence of molybdate (a noncompetitive inhibitor) and phosphate (which exhibits mixed inhibition) results in parallel lines in the reciprocal velocity plots, indicating that phosphate and molybdate compete for a common site; since molybdate is a noncompetitive inhibitor, this site is inferred to be distinct from the site at which substrate binds and is hydrolyzed. Extension of these ideas suggests that phosphate ester substrates should be capable of binding to the molybdate-binding site as well as to the active site, and evidence for substrate inhibition at high substrate concentrations has been obtained. The implications of these findings for interpretation of previous spectroscopic studies of purple acid phosphatase complexes with tetrahedral oxyanions are discussed.

Topics: Acid Phosphatase; Adenosine Monophosphate; Animals; Arsenates; Binding, Competitive; Cattle; Kinetics; Oxidation-Reduction; Phosphates; Phosphotyrosine; Spleen; Substrate Specificity; Tyrosine

The interaction of phosphate with reduced uteroferrin has been re-examined in light of disagreements on the oxidation state of the binuclear iron cluster (Keough, D. T., Beck, J. L., de Jersey, J., and Zerner, B. (1982) Biochem. Biophys. Res. Commun. 108, 1643-1648; Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756). Our results based on Mossbauer observations and the kinetics of spectral change and activity loss show clearly that phosphate binds to reduced uteroferrin to form a reduced uteroferrin-phosphate complex. This complex exhibits a pair of quadrupole doublets at 119 K with parameters typical of a high spin ferric and a high spin ferrous center, respectively, but distinct from those of the native reduced enzyme. The reduced phosphate complex exhibits a pH-dependent visible absorption maximum ranging from 530 to 561 nm. In air, the reduced phosphate complex converts to the oxidized phosphate complex with a first order rate constant of 4 X 10(-3) min-1, as monitored by spectral changes and loss of enzyme activity.

Topics: Acid Phosphatase; Arsenates; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Metalloproteins; Oxidation-Reduction; Phosphates; Spectrophotometry; Tartrate-Resistant Acid Phosphatase