A Study of the Glutathione Transferase Proteome of Drosophila melanogaster: Use of S- Substituted Glutathiones as Affinity Ligands (original) (raw)
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Insect glutathione-S-transferase: A predictor of allelochemical and oxidative stress
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry
The assay conditions for glutathione-S-transferase (GST) 1-chloro-2,4-dinitrobenzene (CDNB) conjugation and cumene hydroperoxide (cumOOH) reduction were optimized in three lepidopteran insect species. 2. The K m for CDNB was 4.1 x 10-4 M, 7.0 X 10-4 M and 2.1 x 10-3 M for Trichoplusia ni, Spodoptera eridania and Papilio polyxenes, respectively. In that order of insect species, the K m for cumOOH was 2.3 x 10-3M, 3.8 x 10-3M and 4.8 x 10-2M. 3. Despite high KmS, the Vma x for both CDNB and cumOOH were high in P. polyxenes, suggesting either a high enzyme concentration or catalytic power represented by the turnover number, Kca t. 4. GST's conjugative activity was in the order of 0.24, 0.09 and 3.19 units in T. ni, S. eridania and P. polyxenes, respectively (1 unit = 1/zmol CDNB conjugated mg protein-I rain-1 at 25°C). 5. The peroxidase activity of GST was high in these insects; 50, 106 and 253 units, respectively, in T. ni, S. eridania and P. polyxenes (I unit = I nmol NADPH oxidized mg protein-I rain-l at 25°C). 6. The high GST's peroxidase activity apparently remedies the minor activity in these insects of the Se-dependent glutathione peroxidase.
Pesticide Biochemistry and Physiology, 2000
Glutathione S-transferase (GST) activity assays in insects are usually performed by spectrophotometric kinetic measurements of conjugated product formation with substrates such as reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). This requires a spectrophotometer that can measure absorbance in the UV range and microcentrifugation to remove the particulates from crude homogenates which absorb light at 340 nm. Such an assay is not ideal for detecting elevated levels of GST activity in insects under field conditions, which is a requirement in, for example, insecticide resistance management programs. We have developed a simple quantitative assay for visually determining GST activity in individual insects. The substrates GSH and CDNB are used in this assay. After the linear enzyme reaction has run for a fixed time, free GSH is determined stoichiometrically by iodometric titration. The results can be determined visually from the discrete color change. We demonstrate the equivalence of this iodometric end point assay and the standard kinetic assay for a five-fold range of purified recombinant Anopheles gambiae agGST1-6 enzyme concentrations and for crude homogenates of individual insects. Results of the application of this test in the diagnosis of GST-based insecticide resistance are presented, demonstrating its practicality for field use.
Insecticide resistance and glutathione S-transferases in mosquitoes: A review
2009
because of their role in insecticide metabolism producing resistance. Many different compounds, including toxic xenobiotics and reactive products of intracellular processes such as lipid peroxidation, act as GST substrates. Elevated levels of GST activity have been reported in organophosphate, organochlorine and pyrethroid resistant mosquitoes. Particulary GST-based resistance is considered the major mechanism of DDT resistance in anopheline species. To date different GST enzymes structurally conserved have been identified suggesting that they may have an important role on common pathways of compound detoxification. In this review we describe the major characteristics of this enzyme family and the principal studies that have contributed to a better knowledge of its role in mosquito insecticide resistance. Finally some aspects on insect GST-based resistance and their implications in traditional biochemical assays for detecting and monitoring GST activity are discussed.
2018
Insecticides are preferred because of their easy applicability in combating pest insects and their short-term results. Insecticides used over dose and unconsciously in agricultural areas cause ecological damage. Also; the use of over dose insecticides in agricultural areas has led to the development of insect resistance. Since the resistance mechanism developed is genetic, it is transmitted to later fertilizers. For this reason, it is important to apply the insecticides in the restricted areas and in the optimal amount. Insects increase their detoxification capacities to protected themselves from insecticides. Detoxification mechanisms are based on the breakdown of insecticides before reaching the target area. The most important detoxification enzyme in insects are Glutathion-S-Transferases (GST). GST enzyme in insects has a role in protection of cellular membranes against oxidative degradation as well as detoxification mechanism. Galleria mellonella may be used as a model organism ...
Bmc Genomics, 2015
Background: Glutathione S-transferases (GSTs) are multifunctional detoxification enzymes that play important roles in insects. The completion of several insect genome projects has enabled the identification and characterization of GST genes over recent years. This study presents a genome-wide investigation of the diamondback moth (DBM), Plutella xylostella, a species in which the GSTs are of special importance because this pest is highly resistant to many insecticides. Results: A total of 22 putative cytosolic GSTs were identified from a published P. xylostella genome and grouped into 6 subclasses (with two unclassified). Delta, Epsilon and Omega GSTs were numerically superior with 5 genes for each of the subclasses. The resulting phylogenetic tree showed that the P. xylostella GSTs were all clustered into Lepidoptera-specific branches. Intron sites and phases as well as GSH binding sites were strongly conserved within each of the subclasses in the GSTs of P. xylostella. Transcriptome-, RNA-seq-and qRT-PCR-based analyses showed that the GST genes were developmental stage-and strain-specifically expressed. Most of the highly expressed genes in insecticide resistant strains were also predominantly expressed in the Malpighian tubules, midgut or epidermis. Conclusions: To date, this is the most comprehensive study on genome-wide identification, characterization and expression profiling of the GST family in P. xylostella. The diversified features and expression patterns of the GSTs are inferred to be associated with the capacity of this species to develop resistance to a wide range of pesticides and biological toxins. Our findings provide a base for functional research on specific GST genes, a better understanding of the evolution of insecticide resistance, and strategies for more sustainable management of the pest.
Mosquito glutathione transferases
Methods in enzymology, 2005
The glutathione transferases (glutathione S-transferases, GSTs) are a diverse family of enzymes involved in a wide range of biological processes, many of which involve the conjugation of the tripeptide glutathione to an electrophilic substrate. Relatively little is known about the endogenous substrates of mosquito GSTs, and most studies have focused on their role in insecticide metabolism, because elevated levels of GST activity have been associated with resistance to all the major classes of insecticides. In addition, there is growing interest in the role of this enzyme family in maintaining the redox status of the mosquito cell, particularly in relation to vectorial capacity. Most GSTs are cytosolic dimeric proteins, although a smaller class of microsomal GSTs exists in insects, mammals, and plants. Each GST subunit has a G site that binds glutathione and a substrate-binding site or H site. There are more than 30 GST genes in mosquitoes. Additional diversity is contributed by alte...
Annals of the Entomological Society of America, 2004
The Hessian ßy, Mayetiola destructor (Say), poses a signiÞcant economic threat to wheat in terms of reduced grain yield, particularly in the eastern soft-winter-wheat region of the United States. However, little is know about the molecular mechanisms involved in the plantÐinsect interaction. The glutathione S-transferases (GSTs) form a large family of enzymes that protect cells from damage by reactive electrophilic compounds. We have cloned and characterized two GST genes from biotype GP of the Hessian ßy. Sequence analysis and homology searches of the coding region for the Þrst gene (designated mdesgst1-1) indicated it contained an intact coding region for a GST-like protein sharing homology to the insect class I GSTs. Analysis of the coding region for the second gene indicated it contained numerous stops and was most probably a pseudogene. Southern analysis and in situ hybridization on polytene chromosomes suggested the class I GSTs in M. destructor are encoded by a small gene family that is arranged sequentially on the short-arm of chromosome X1. Expression in vitro of the protein encoded by mdesgst1-1, and biochemical analysis of activity conÞrmed the mdesGST1-1 protein was catalytically active. Reverse transcription-polymerase chain reaction revealed mdesgst1-1 was expressed in midgut tissue, fat body, and salivary glands of larvae. Preliminary results from double-stranded RNA interference of GST gene expression seem to suggest a role for GSTs in the biochemical adaptation of M. destructor larvae to wheat.
The combination of proteomic approach and affinity chromatography (GSTrap™ HP) has been used to identify and investigate the differential expression of GSTs under toxic challenge and during the course of development in Aedes albopictus. There were multiple isoforms of glutathione transferases (GSTs) expressed in the three developmental stages of Aedes albopictus. On a 2D gel map, the results indicate that there were 19 spots with a molecular mass in the range of 24-30 kDa. Three types of GSTs were identified from the spots; namely GSTS1, GSTD1, and GST Theta. Spots identified as GSTS1 and GSTD1 were further designated as GSTS1-1 to GSTS1-5 and GSTD1-1 to GSTD1-13 respectively. Isoforms GSTD1-10, GSTD1-11, GSTD1-12, GSTD1-13, and GSTT were absent in the adult life stage. Isoenzymes GSTS1-3, GSTD1-2, and GSTD1-6 appeared as the major expressed isoforms in all the life stages. The effect of free radical generators such as hydrogen peroxide and paraquat on the expression of the GST isoforms in larvae mosquitoes was also investigated. Isoforms GSTD1-1, GSTD1-4, GSTS1-4, and GSTD1-12 had N1.5 fold increase when the larvae were challenged with both chemicals. GSTS1-3, GSTS1-4, GSTD1-1, GSTD1-2, GSTD1-3, GSTD1-6, and GSTD1-12 were significantly expressed (p b 0.05) with N1.5 fold change when the larvae were challenged with hydrogen peroxide. On the contrary, different GST isoforms were responsive towards paraquat challenge. Those isoforms were GSTS1-2, GSTS1-4, GSTD1-1, GSTD1-5, GSTD1-9, GSTD1-11, GSTD1-12, and GSTT (with N 1.5 fold change). The purified larvae GSTs seemed to have limited substrate specificities with the ability to only conjugate 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB). The GSTs have however shown that they were capable of detoxifying lipid peroxidation products and possessing glutathione peroxidase activities.