Differential uptake of mercury vapor by gramineous c(3) and c(4) plants

The uptake of mercury vapor by six gramineous plant species was compared under uniform conditions using a whole-plant chamber and ²⁰³Hg-labeled mercury at a low atmospheric concentration. Mean Hg uptake by leaves of the C₃ species oats (Avena sativa), barley (Hordeum vulgare), and wheat (Triticum aestivum) was 5 times greater than that by leaves of the C₄ species corn (Zea mays), sorghum (Sorghum bicolor), and crabgrass (Digitaria sanguinalis). Although there was a difference in resistances associated with vapor entry into the leaves, as shown by estimates of gas exchange, the differential uptake by C₃ and C₄ species was largely attributable to internal resistances to Hg vapor binding. The nature of the internal resistances and the site or sites of Hg vapor binding remain unspecified.     

Properties of leaf NAD malic enzyme from plants with C4 pathway photosynthesis

C₄ acid decarboxylation in one group of C₄-pathway species is mediated by an NAD malic enzyme. This paper reports on the partial purification and properties of this enzyme from three species of this group, Atriplex spongiosa, Amaranthus edulis, and Panicum miliaceum. Depending upon the conditions, the Atriplex spongiosa enzyme was 5–30% as active with NADP compared with NAD but the enzyme from the other species was specific for NAD. The enzyme from each species had an absolute requirement for Mn²⁺ that could not be replaced by Mg²⁺, and activity was increased several fold by low concentrations of either CoA or acetyl CoA. For the enzyme from Atriplex spongiosa and Amaranthus edulis, there was cooperativity for malate binding and the activators CoA and acetyl CoA functioned to increase the affinity of malate for the enzyme. The Hill coefficients for malate binding were approximately 2 and 4, respectively. However, with the enzyme from Panicum miliaceum, cooperative binding of malate was not apparent and activators operated by increasing V rather than the affinity for malate. Bicarbonate inhibited the enzyme from Atriplex spongiosa and Amaranthus edulis and its effect was inversely related to the concentrations of malate, NAD, and activators. The possible significance of these various allosteric effects on the regulation of the enzyme in vivo is discussed. Reactant concentrations and other conditions required for maximum activity are reported.     

Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : III. Reduction of Photorespiration by a Limited C(4) Pathway of Photosynthesis in Flaveria ramosissima

The initial products of photosynthesis by the C₃ species Flaveria cronquistii, the C₄ species F. trinervia, and the C₃-C₄ intermediate species F. ramosissima were determined using a pulse-chase technique with ¹⁴CO₂-¹²CO₂. The intermediate species F. ramosissima incorporated at least 42% of the total soluble ¹⁴C fixed into malate and aspartate after 10 seconds of photosynthesis in ¹⁴CO₂, as compared with 90% for the C₄ species F. trinervia and 5% for the C₃ species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in ¹²CO₂, although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C₄ pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C₃ and C₄ plants.     

Functional binding of hexanucleotides to 3C protease of hepatitis A virus

Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C^pro). Inhibition assays in vitro identified the hexanucleotide 5′-GGGGGT-3′ (G₅T) as a 3C^pro protease inhibitor. Using ¹H NMR spectroscopy, G₅T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of ¹H, ¹⁵N-HSQC experiments the binding site for G₅T was located to the C-terminal β-barrel of HAV 3C^pro. Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G₅T-binding site, nor does G₅T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid–protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.     

Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C₆, C₁₂, C₁₆, C_18:1, and C_24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe²⁺. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.     

Alishewanella solinquinati sp. nov., Isolated from Soil Contaminated with Textile Dyes

A novel Gram-negative, motile, rod-shaped, facultative anaerobic bacterial strain, KMK6^T, was isolated from soil contaminated with textile dyes from an industrial estate located at Ichalkaranji, Maharashtra, India, and its taxonomical position was established by using a polyphasic approach. The major cellular fatty acids included C_17:1ω8c, summed feature 3 (C_16:1ω7c and/or iso-C_15:0 2-OH), C_17:0, C_16:0, and C_18:1ω7c. The DNA G+C content of strain KMK6^T was 48.8 mol %. 16S rRNA gene sequence analysis confirmed its placement in the genus Alishewanella, and exhibited sequence similarity levels of below 97 % to the type strains of validly published Alishewanella species. On the basis of genotypic and phenotypic evidence, strains KMK6^T is considered to be a novel species of the genus Alishewanella, for which we propose that strain KMK6^T (=NCIM 5295^T =BCRC 17848^T) is assigned to a novel species, Alishewanella solinquinati sp. nov.     

Responses of the carbon and oxygen isotope compositions of desert plants to spatial variation in soil salinity in Central Asia

We examined the isotopic parameters in two C₃ species (Artemisia diffusa H. Krasch and Tamarix hispida Willd.) and a C₄ species [Haloxylon aphyllum (Minkw.) Iljin.] growing or planted in soils with different levels of salinity in a Central Asian desert. The oxygen isotope ratios of stem water (δ¹⁸O_stem) in T. hispida and H. aphyllum distributed in high-salinity zones were similar to the δ¹⁸O of artesian water (δ¹⁸O_artesian) and different from that in A. diffusa distributed in lower-salinity zones. This indicates that T. hispida and H. aphyllum depend on water with low salinity in the deeper soil layer, whereas A. diffusa depends on water in the shallower soil layer that would be affected by salt accumulation. The carbon isotope composition of leaf organic matter (δ¹³C_om) and oxygen isotope enrichment in leaf organic matter above stem water (Δ¹⁸O_om) were lower in A. diffusa than in the other species. The responses of δ¹³C_om and Δ¹⁸O_om to soil salinity observed for T. hispida suggest that the species decreased its transpiration rate and increased its intrinsic water-use efficiency in response to increasing soil salinity. The δ¹³C_om and Δ¹⁸O_om of H. aphyllum were higher than those of the C₃ species, and were not correlated with soil salinity, suggesting that H. aphyllum reduced its salt uptake by decreasing transpiration—even though it was able to access less saline water in the deeper soil layer. These results indicate that the water-use strategy of desert plants in high-salinity environments can be assessed based on their carbon and oxygen isotope ratios.     

Peptide NMHRYPNQ of the Cellular Prion Protein (PrP) Inhibits Aggregation and Is a Potential Key for Understanding Prion-Prion Interactions

Pathogenesis of transmissible spongiform encephalopathies is correlated with a conversion of the normal cellular form of the prion protein (PrP^C) into the abnormal isoform (scrapie form of PrP). Contact of the normal PrP with its abnormal isoform, the scrapie form of PrP, induces the transformation. Knowledge of molecules that inhibit such contacts leads to an understanding of the mechanism of the aggregation, and these molecules may serve as leads for drugs against transmissible spongiform encephalopathies. Therefore, we screened a synthetic octapeptide library of the globular domain of the human PrP^C for binding affinity to PrP^C. Two fragments with binding affinity, 149YYRENMHR156 and 153NMHRYPNQ160, were identified with K_d values of 21 and 25 μM, respectively. A 10-fold excess of peptide 153NMHRYPNQ160 inhibits aggregation of the PrP by 99%. NMR and mass spectrometry showed that the binding region of the peptide 153NMHRYPNQ160 is located at helix 3 of the PrP.     

Cation binding to beta-casein. A comparison of electrostatic models

The binding of cations of β-casein at pH 6.6 was considered previously. Available for three sodium concentiations, I = 0.04, 0.08, or 0.16 M are: [1] proton releases between I and [2] for each I, as calcium activity is increased, correlated sequences of monomer net charge, proton release, site bound calcium and protein Solvation- Models for ion binding are examined. Critical considerations are the intrinsic binding constants between hydrogen[H], calcium[Ca] and sodium[Na] ions and phosphate[P] and caiboxyIate[C] sites, and the effects of electrostatic interaction between sites as influenced by spatial fixed charge distribution, ionic strength and dielectric constant [D]. Anticipated intrinsic binding constants are k_H,P^o = 3 × 10⁶, k_Ca,P^o = 120, k_Na,P^o = 1, k_H,C^o = 7 × 10⁴ and k_Ca,C^o = 5.6Distributed charge models, either surface or volume, are inadequate since any reasonable monomer size yields fixed charge densities requiring k_H,P^o and k_Ca,C^o which are too low when the maximum in D is 75. Also, with increasing calcium binding, calculated proton release is only 0.4 to 0.5 of that observed.Discrete charge models accept anticipated k^o and yield calculated sequences of calcium binding and proton release which are in good agreement with those observed provided that: (1) using the known amino acid sequence of the phosphate-containing acidic peptide portion of the molecule, pep tide fixed charge is distributed at the lowest I so as to minimize electrostatic free energy; (2) in the region of fixed charge, D is approximately 5; (3) the distances between peptide fixed charges decrease with increasing ionic strength or calcium binding and (4) while protein is in solution, the acidic peptide and the remainder of the molecule are essentially electrostatically independent.     

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRAS^G12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS^G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK_a of the C12 thiol is depressed (pK_a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS^Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS^G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K_d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK_a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS^G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS^G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS^G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS^G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.     

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : I. CO(2) Assimilation and Metabolism and Activities of Phosphoenolpyruvate Carboxylase and NADP-Malic Enzyme

The degree of C₄ photosynthesis was assessed in four hybrids among C₄, C₄-like, and C₃-C₄ species in the genus Flaveria using ¹⁴C labeling, CO₂ exchange, ¹³C discrimination, and C₄ enzyme activities. The hybrids incorporated from 57 to 88% of the ¹⁴C assimilated in a 10-s exposure into C₄ acids compared with 26% for the C₃-C₄ species Flaveria linearis, 91% for the C₄ species Flaveria trinervia, and 87% for the C₄-like Flaveria brownii. Those plants with high percentages of ¹⁴C initially fixed into C₄ acids also metabolized the C₄ acids quickly, and the percentage of ¹⁴C in 3-phosphoglyceric acid plus sugar phosphates increased for at least a 30-s exposure to ¹²CO₂. This indicated a high degree of coordination between the carbon accumulation and reduction phases of the C₄ and C₃ cycles. Synthesis and metabolism of C₄ acids by the species and their hybrids were highly and linearly correlated with discrimination against ¹³C. The relationship of ¹³C discrimination or ¹⁴C metabolism to O₂ inhibition of photosynthesis was curvilinear, changing more rapidly at C₄-like values of ¹⁴C metabolism and ¹³C discrimination. Incorporation of initial ¹⁴C into C₄ acids showed a biphasic increase with increased activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme (steep at low activities), but turnover of C₄ acids was linearly related to NADP-malic enzyme activity. Several other traits were closely related to the in vitro activity of NADP-malic enzyme but not phosphoenolpyruvate carboxylase. The data indicate that the hybrids have variable degrees of C₄ photosynthesis but that the carbon accumulation and reduction portions of the C₄ and C₃ cycles are well coordinated.     

The β Subunit of Eukaryotic Translation Initiation Factor 2 Binds mRNA through the Lysine Repeats and a Region Comprising the C2-C2 Motif

Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNA^Met have been identified. All such mutations in the beta subunit of eIF2 (eIF2β) mapped to a region containing a putative zinc finger structure of the C₂-C₂ type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2β that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2β, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C₂-C₂ motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2β, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNA^Met binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.     

Photosynthetic activities of isolated bundle sheath cells in relation to differing mechanisms of C-4 pathway photosynthesis.

Photosynthetic activities of bundle sheath cell strands isolated from several C₄ pathway species were examined. These included species that decarboxylate C₄ acids via either NADP-malic enzyme (Zea mays, NADP-malic enzyme-type), NAD-malic enzyme (Atriplex spongiosa and Panicum miliaceum, NAD-malic enzyme-type) or phosphoenolpyruvate carboxykinase (Chloris gayana and Panicum maximum, phosphoenolpyruvate carboxykinase-type). Preparations from each of these species fixed ¹⁴CO₂ at rates ranging between 1.2 and 3.5 μmol min^?1 mg^?1 of chlorophyll, with more than 90% of the ¹⁴C being assimilated into Calvin cycle intermediates. With added HCO₃^? the rate of light-dependent O₂ evolution ranged between 2 and 4 μmol min^?1 mg^?1 of chlorophyll for cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species but with Z. mays cells there was no O₂ evolution detectable. Most of the ¹⁴CO₂ fixed by Z. mays cells provided with H¹⁴CO₃^? plus ribose 5-phosphate accumulated in the C-1 of 3-phosphoglycerate. However, 3-phosphoglycerate reduction was increased several fold when malate was also provided. Cells from all species rapidly decarboxylated C₄ acids under appropriate conditions, and the CO₂ released from the C-4 carboxyl was reassimilated via the Calvin cycle. Malate decarboxylation by Z. mays cells was dependent upon light and an endogenous or exogenous source of 3-phosphoglycerate. Bundle sheath cells of NAD-malic enzyme-type species rapidly decarboxylated [¹⁴C]malate when aspartate and 2-oxoglutarate were also provided, and [¹⁴C]aspartate was decarboxylated at similar rates when 2-oxoglutarate was added. Cells from phosphoenolpyruvate carboxykinase-type species decarboxylated [¹⁴C]aspartate when 2-oxoglutarate was added and they also catalyzed a slower decarboxylation of malate. Cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species evolved O₂ in the light when C₄ acids were added. These results are discussed in relation to proposed mechanisms for photosynthetic metabolism in the bundle sheath cells of species utilizing C₄ pathway photosynthesis.     

Effects of O(2) Tension and Temperature during Light Uptake of CO(2) on the Dark Release of CO(2) by Excised C(3) and C(4) Leaves

A study was conducted on a C₄ (Panicum maximum) and a C₃ (Panicum bisulcatum) species to determine the nature of the dark release of ¹⁴CO₂ with respect to its responses to changes in temperature and O₂ tension during light CO₂ uptake of ¹⁴CO₂.     

Structural Basis for Difference in Heat Capacity Increments for Ca Binding to Two α-Lactalbumins

Thermodynamic parameters for the unfolding of as well as for the binding of Ca²⁺ to goat α-lactalbumin (GLA) and bovine α-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25°C. Among the different parameters available, the heat capacity increments (ΔC_p) offer the most direct information for the associated conformational changes of the protein variants. The ΔC_p values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective ΔC_p values for the binding of Ca²⁺ are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca²⁺-dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca²⁺-binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in ΔC_p values for the binding of Ca²⁺ is presumably in part related to this difference in charge distribution.     

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C₈-acyl carrier protein (C₈-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4′-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C₈-CoA. Furthermore, our ¹H saturation transfer difference and ³¹P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved ⁷¹RGG⁷³ loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.     

Crystal structure of the C-terminal region of Streptococcus mutans antigen I/II and characterization of salivary agglutinin adherence domains

The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 Å resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C₁, C₂, and C₃. Each domain adopts a DE-variant IgG fold, with two β-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C₁ and C₂) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C₁ and C₂ domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C₁ and C₂ domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.     

Protein-lipoprotein interactions in the haemolymph of Locusta during the action of adipokinetic hormone: The role of CL-proteins

The involvement of C_L-proteins in the formation of lipoprotein A⁺ during adipokinetic hormone action has been investigated using radiolabelling experiments. Injected [³H]-C_L-proteins associate rapidly with lipoprotein A⁺ during its formation. Both [³H]-C_L-proteins and [³H]-Ayellow are liberated from [³H]-A⁺ during its natural degradation in the haemolymph (when adipokinetic hormone action is declining). It appears that [³H]-C_L-proteins bind reversibly to A⁺, since they are easily displaced in vivo and in vitro by competing concentrations of non-labelled C_L-proteins. It is suggested that Ayellow is an integral component of the A⁺ lipoprotein complex, whereas C_L-proteins may play only a relatively minor part in its structural organisation. Possible functions of the binding of C_L-proteins to A⁺ are discussed.     

Survey of pyruvate,phosphate dikinase activity of plants in relation to the C3, C4 and CAM mechanisms of CO2 assimilation

The pyruvate, phosphate dikinase activity (PPD, EC 2.7.9.1) associated with crude extracts of leaf tissue of some C₃ and C₄ plants was determined by phosphoenolpyruvate plus PPi-dependent phosphorylation of AMP. The PPD activity of all C₄ plants examined was > 15 nmol/mg protein/min. Several factors contributed to the underestimation of PPD activity in crude extracts of at least some species. Significant PPD activity (> 0.15 nmol/mg protein/min) was not detected in the majority of C₃ species but several C₃ species and the two CAM species studied exhibited activity in the range 0.4–4 nmol/mg protein/min while the C₃ species Avena sativa showed activity up to 8 nmol/mg protein/min. The oat leaf enzyme was partially purified; it exhibited properties similar to those of partially purified PPD from maize. Leaf extracts of the orchids Cymbidium canaliculatum and C. madidum contained high levels of PPD activity similar to the majority of C₄ plants. PPD activity has also been shown in other previously unstudied species.     

Physiological and antioxidant enzyme responses to acute and chronic exposure of Laeonereis acuta (Polychaeta, Nereididae) to copper

Chronic (14 days) and acute (48 h) copper effects on the antioxidant defense system and some physiological variables of Laeonereis acuta (Polychaeta, Nereididae) were evaluated. In both assays, two nominal copper concentrations (chronic: C₁=31.25 and C₂=62.50 μg/l; acute: A₁=250 and A₂=500 μg/l) and one control group (C_c and A_c=0 μg/l) were tested. End points analyzed were antioxidant enzyme activity (catalase, CAT; superoxide dismutase, SOD; and glutathione S-transferase, GST), oxygen consumption, metahemoglobin concentration, and lipid peroxidation (LPO). In the chronic assay, CAT activity was significantly higher in worms exposed to both concentrations of copper tested (C₁=3.36±0.07 U CAT/mg protein; C₂=4.06 0.32 U CAT/mg protein) than in control worms (C_c=2.16±0.39 U CAT/mg protein). SOD activity was also increased in the two copper-exposed groups (C₁=16.85±4.22 U SOD/mg protein; C₂=38.19±4.31 U SOD/mg protein) than in control group (C_c=3.54±0.46 U SOD/mg protein). However, GST activity was increased only in worms exposed to the higher copper concentration (C₂=0.022±9.10⁻⁴ U GST/mg protein) when compared to the other groups tested (C_c=0.012±3.10⁻³ U GST/mg protein; C₁=0.016±9.10⁻⁴ U GST/mg protein). None of the physiological variables analyzed (oxygen consumption, metahemoglobin concentration, and lipid peroxidation) was affected by chronic copper exposure. In the acute assay, only GST activity was induced in worms exposed to copper. This induction was observed only in the A₁ group (0.027±2.10⁻³ U GST/mg protein) when compared to A_c (0.017±2.10⁻³ U GST/mg protein) or A₂ (0.016±7.10⁻⁴ U GST/mg protein) groups. On the other hand, lipid peroxidation was higher in A₂ (481.9±49.2 nmol CHP/g ww) than in control worms (A_c=337.9±25.0 nmol CHP/g ww). Oxygen consumption was higher in worms acutely exposed to the lower copper concentration tested (A₁=0.27±0.04 mg O₂/g ww/h) than in the higher concentration (A₂=0.14±0.01 mg O₂/g ww/h). Changes in the swimming behavior of copper-exposed animals in both assays and edemas in the body wall of worms acutely exposed to copper were also observed. Results suggest that copper exposure favors reactive oxygen species generation and that enzymatic defense system is induced under chronic exposure, preventing oxygen consumption changes and lipid peroxidation and metahemoglobin formation. However, in acutely exposed worms, in spite of a transient peak of GST activity, no induction of antioxidant enzymes occurs, leading to morphological and physiological changes.