Purification and characterization of phenoloxidase from clam Ruditapes philippinarum

Using L-dihydroxyphenylalanine (L-DOPA) as a specific substrate, phenoloxidase (PO) from clam (Ruditapes philippinarum) was purified by Q Sepharose Fast Flow ion-exchange chromatography and Sephacryl S-100 gel-filtration, and characterized biochemically and enzymatically in this study. The molecular mass of PO in SDS-PAGE is about 76.9 kDa, and the prophenoloxidase (proPO) molecule, isolated as a monomeric protein, is 84.1 kDa. The PO molecule had a high oxidative activity, and the proPO molecule had almost no oxidative activity. The PO activity was optimal at pH 7.0 and temperature of 40 degrees C. The Km value of the PO for L-DOPA was 2.2 mmol l(-1). The PO was extremely sensitive to benzoic acid and sodium sulfite, very sensitive to citric acid, thio urea, 1-phenyl-2-thiourea and cysteine, but not sensitive to ascorbic acid. Combined with its specific enzyme activity on tyrosine and L-DOPA, it can be concluded that the Ruditapes PO is probably a kind of tyrosinase-type phenoloxidase. The PO activity was strongly inhibited by ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DETC), Zn2+, Ca2+ and Cu2+, as well as by Mg2+. The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu2+ on DETC-inhibited PO activity, indicate that Ruditapes PO is most probably a copper-containing metalloenzyme.     

Purification and partial characterization of the glutathione S-transferase of rat erythrocytes

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (M_r 48 000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of M_r 23 500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The K_m values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I₅₀) were as follows: 12 μM bromosulphophthalein, 34 μM S-hexylglutathione, 339 μM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (K_i = 8 μM) and glutathione (K_is = 4 μM; K_ii = 11.5 μM) while S-hexylglutathione was competitive with glutathione (K_i = 5 μM).     

Spatial and seasonal biomarker responses in the clam Ruditapes decussatus

The clam Ruditapes decussatus is an important resource to preserve in coastal lagoon systems around the Mediterranean including the South Portugal. To assess spatial and temporal biomarker responses to contamination in the species, a multibiomarker approach was conducted using antioxidant enzymes, MFO system phase I and II; acetylcholinesterase, metallothionein (MT), δ-aminolevulinic acid dehydratase and lipid peroxidation (LPO). The condition index (CI), metals and polycyclic aromatic hydrocarbons (PAHs) were also determined. The levels of contaminants were not particularly high and the antioxidant enzymes, acetylcholinesterase (AChE), MT in the digestive gland, and δ-aminolevulinic acid dehydratase (ALAD) do not provide a suitable seasonal and spatial discrimination reversely to that regarding CYP450, glutathione-S-transferase (GST), MT in the gills, and LPO in both tissues. However, even those could vary with natural variables that may act as confounding factors. Thus, seasonal variability and natural range of biomarker responses must be carefully and accurately taken into account in ecotoxicological approaches of environmental quality assessment programmes.     

Purification and characterization of corn glutathione S-transferase

Two glutathione S-transferase (GST) activities have been identified and purified from etiolated corn tissue. The first, designated GST I enzyme, is constitutively present in corn tissue, and the second, designated GST II enzyme, is present only in tissue which has been treated with chemical antidotes which protect corn against chloroacetanilide herbicides. The total activity constitutes approximately 2% of the soluble protein in these tissues. The native forms of these enzymes have molecular weights of approximately 50 000 as determined by Sephadex G-100 chromatography. On sodium dodecyl sulfate-polyacrylamide gels, GST I enzyme migrates primarily as a single band of molecular weight 29 000, and GST II enzyme migrates as primarily two bands of molecular weight 29 000 and 27 000. Both enzymes catalyze the formation of a glutathione-herbicide conjugate in vitro when the herbicide alachlor is used as a substrate. This conjugation results in elimination of the biological activity of the herbicide.     

Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae.

Glutathione S-transferase (GST) has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by Chironomidae larvae. We have purified and characterized GST from cytosolic fractions of Chironomidae larvae. GST with an M(r) of 23 kDa has been purified to homogeneity from larvae by centrifugation, size exclusion chromatography on Sephadex G25, and glutathione affinity and anion exchange chromatography. The purified enzyme exhibited moderate activity towards 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxide, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 5.5. The enzyme had a maximum activity at approximately pH 8 and showed activity between 30 and 40 degrees C. It became inactive at higher temperature (>50 degrees C) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of conserved regions in the enzyme. The enzyme activity was comparable to levels of metabolism observed by animal GST involved in the detoxification of xenobiotics.     

Virus-like particles associated with mortalities of the carpet-shell clam Ruditapes decussatus

During the late summer and early fall of 1997 and 1998 heavy mortalities were detected in the carpet-shell clam Ruditapes decussatus cultured in Galicia (NW Spain). The prevalences and intensities of the parasites found in the histopathological studies were not high enough to explain the high mortality rates. Unenveloped virus-like particles were detected by transmission electron microscopy in the cytoplasm of the connective tissue cells. They had an icosahedrical-spherical shape with a diameter of 27 to 35 nm. These virus-like particles appeared free in the cytoplasm or associated with endoplasmic reticulum membranes. These characteristics suggest that they belong to the Picornaviridae family.     

Purification and characterization of camel liver glutathione S-transferase

• 1.1. Glutathione S-transferases have been purified (18-fold) in 65–70% yield from the liver of one humped camel using affinity chromatography on glutathione-linked agarose.
• 2.2. Chromatofocusing technique resolves the glutathione S-transferases into seven distinct isoenzymes with apparent pI of 8.7, 8.4, 8.0, 7.8, 7.3 and 6.5.
• 3.3. The major isoenzyme (pI 8.7) which accounted for over 95% of the total activity was composed of two identical subunits of molecular mass 24,000 and was immunologically similar to the other six isoenzymes.
• 4.4. The substrate specificities and the effect of various inhibitors on the activity of the abundant camel liver isoenzyme were also examined.

     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

Purification and characterization of glutathione S-transferase isozymes in dog lens.

1. Two isozymes of glutathione S-transferase (GST-dl1 and GST-dl2) were purified to homogeneity from dog lens. 2. The subunit size and the isoelectric point were determined to be 24,000 and > pI 9.5 for GST-dl1 and 22,000 and pI 8.1 for GST-dl2. 3. It was judged that GST-dl1 is a class alpha enzyme and GST-dl2 belongs to class pi on the basis of their immunological properties and N-terminal amino acid sequences. 4. The expression pattern of glutathione S-transferase isoenzymes in dog lens is different from that in pig, rat and bovine lenses.     

Molecular detection of a haplosporidian parasite in carpet shell clam Ruditapes decussatus from Spain

A protozoan parasite identified by histology as a haplosporidian was detected in carpet shell clam Ruditapes decussatus from Spain during routine samplings. Analysis based on the sequence of the small subunit ribosomal RNA gene revealed that this organism was related to the haplosporidian group, mainly to Urosporidium. A specific probe was used for in situ hybridization studies to show the specificity of the amplified sequence.     

Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver.

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].     

Differentially expressed genes of the carpet shell clam Ruditapes decussatus against Perkinsus olseni

Suppression–Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a reverse subtraction were carried out to identify up- and down-regulated genes in both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for further investigation into the functional basis of resistance to pathogens, have been detected for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of ESTs with potential similarity with known genes was 97, 42 among them were related with immunity and stress related functions. The pattern of expression of the immune selected genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams and compared with samples from an in vitro infection of clam haemocytes with Perkinsus zoospores. The maximum expression was found 1 h post infection. The complete open reading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined. Our results provide new insights into the molecular basis of host–pathogen interactions in R. decussatus.     

Morphological characterization and functional immune response of the carpet shell clam (Ruditapes decussatus) haemocytes after bacterial stimulation

The morphology and functionality of Ruditapes decussatus haemocytes have been characterized by light microscopy and flow cytometry, leading to the identification of three different cellular subpopulations. Granulocytes were the largest cells, the hyalinocytes were smaller and contained fewer granules and the intermediate cells showed a size similar to hyalinocytes and a higher number of granules. The phagocytosis of different particles and the associated production of oxygen radicals were measured by flow cytometric methods. Granulocytes were the most active cells, followed by the intermediate cells and hyalinocytes. The effect of stimulation of haemocytes with lipopolysaccharide (LPS), with a heat inactivated bacterial mixture or with the infection of Vibrio splendidus on the cell viability and the expression of selected immune-related genes were studied. While significant low levels of damaged cells were registered in LPS-stimulated cells, the treatment with dead bacteria or V. splendidus reduced cell viability 1 h, 3 h and 6 h after treatment. The stimulation of haemocytes with LPS and dead bacteria induced changes in the expression of defender against cell death (DAD-1), thrombin, prosaposin, inhibitor of apoptosis (IAP), factor B and C3 complement component.     

Purification and characterization of a new form of glutathione S-transferase from human erythrocytes

Presence of a new form of glutathione S-transferase has been demonstrated in human erythrocytes. using two different affinity ligands this enzyme has been separated from the previously characterized glutathione S-transferases ?. The new enzyme is highly basic with a pI of > 10. The new enzyme which represents less than 5 percent of glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene as substrate and about 10 percent of total glutathione S-transferase protein of erythrocytes has different amino acid composition, substrate specificities, and immunological characteristics from those of the major erythrocyte glutathione S-transferase ?. Immunological properties of the new enzyme indicate that this form may be different from other glutathione S-transferases of human tissues.     

Purification and partial characterization of glutathione transferase from the teleost Monopterus albus

Glutathione transferases (GSTs) catalyze the transfer of glutathione to a variety of xenobiotic and toxic endogenous compounds. GSTs are phase II biotransformation enzymes and are proposed as biomarkers of environmental pollution. In this study, a cytosolic glutathione transferase (maGST) was purified from liver of the freshwater fish Monopterus albus by affinity chromatography. The maGST appeared to be a homodimer composed of two subunits each with a molecular weight of 26 kDa. This maGST showed high activity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with CDNB as substrate revealed a K(m) of 0.28 mM and V(max) of 15.68 micromol/min per mg of protein. It had maximum activity in the pH range 7.0-7.5, a broad optimum T(m) range of 30 degrees C-55 degrees C, and a high thermal stability with 77% of its initial activity at 45 degrees C. This high thermal stability of maGST could be related to the physiological adaptation of M. albus to high temperatures in tropical and subtropical environments.     

可食用文蛤中氧化性胁迫的生物标记物与甲基毒死蜱和草甘膦的生物富集

近几十年期间,在种群和个体受干扰后,对作为早期指示剂的生物标记物的研究受到越来越多的关注。我们用对水生生态系统污染敏感的生物标记物双壳类软体动物(文蛤)来评估两种有机磷杀虫剂(甲基毒死蜱、草甘膦)的影响。文蛤是水生生态系统污染的一种敏感的指示物种。在不同时间段测定文蛤中不同组织的非酶的(谷胱苷肽)和酶的(过氧化氢酶)抗氧化剂,作为文蛤中生物标记物的反应。在实验室条件下,测定了脂质过氧化作用、蛋白羰基含量、总蛋白含量、总脂质含量以及胆碱酯酶的活性。对不同的生物标记与杀虫剂的生物体内积累的相互关系进行了研究。甲基毒死蜱在文蛤组织中具有最大的诱导氧化胁迫的潜能,导致脂质过氧化反应增加并抑制抗氧化剂。而且,鳃是对该反应最敏感的器官。文蛤是一种极好的甲基毒死蜱的积聚者,因为暴露60天后,可以测定到其组织中浓度为824.0 mg/kg w.w的甲基毒死蜱。随着在草甘膦中暴露时间的增加,与背景水平相比,组织中草甘膦的浓度增加大约8×103mg/kg w.w。可以得出这样的结论:在一种生物中测定几种生物标记物是有用的。在双壳类中,蛋白质的羰基诱导可用于双壳类中化学污染物诱导的氧化胁迫的生物指示剂。抗氧化剂的防御成分是敏感的参数,是评估污染的水生生态系统的有用的生物标记物。辅以蛤组织的化学分析,生物标记参数能够提供一种有力的监测工具。     

Purification and partial characterization of glutathione S-transferase from insecticide-resistant field populations of Liposcelis paeta Pearman (Psocoptera: Liposcelididae)

Enzymes that possess glutathione S-transferase (GST) activity were purified to homogeneity by glutathione-agarose affinity chromatography from three field populations of Liposcelis paeta (Pearman). These populations were collected from Nanyang city of Henan Province (NY), Wuzhou (WZ) and Hezhou (HZ) cities of Guangxi Province, China, and had different susceptibilities to dichlorvos [LC₅₀s of the NY (281.48 mg/m²), the WZ (285.07 mg/m²), and the HZ (243.52 mg/m²), respectively]. The specific activities of purified enzymes from these three populations increased 32.24-, 99.81-, and 42.52-fold, respectively. Kinetic analyses showed that the catalytic activity of purified GST from NY population towards GSH was much higher than the others, while WZ population reached the highest in V. SDS–polyacrylamide electrophoresis revealed that the purified GST had two subunits with a molecular mass of 23.31 and 20.43 kDa for NY, 53.14 and 20.13 kDa for WZ, and 50.79 and 19.42 kDa for HZ, respectively. The in vitro inhibition studies of GSTs indicated that three kinds of insecticides (chlorpyrifos, carbosulfan, and cypermethrin) and five metallic ions (Zn²⁺, Ba²⁺, Ca²⁺, Hg²⁺, Mn²⁺, and Mg²⁺) all possessed inhibitory effects on purified GST, and ethacrynic acid (EA, a specific inhibitor of GST) expressed inhibitory effects. In the bioassay, three populations of L. paeta had different susceptibilities to different insecticides, even after they were reared on diets consisting of 25% EA. The GST activities of L. paeta from different areas also showed different temperature and pH stabilities. The differences in GST among the three populations may be attributed partially to the differences in control practices for psocids between Henan and Guangxi Provinces. © 2009 Wiley Periodicals, Inc.