Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification

In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; M_r 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to M_r 52000 (PLAT Y₁) and M_r 45000 (PLAT Y₂) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y₂ had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y₁- and PLAT Y₂-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y₂ accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y₁ and PLAT Y₂ fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y₁ and PLAT Y₂ for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y₁ and PLAT Y₂. The major glutathione S-transferase from the PLAT Y₂ fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y₂-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.     

Glutathione S-Transferase Isoenzymes from Streptomyces griseus

An inducible, cytosolic glutathione S-transferase (GST) was purified from Streptomyces griseus. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native M_rs of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.     

Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or “wild-type” velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K_m values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V_max for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V_max for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.     

Glutathione transferase from Plasmodium falciparum – Interaction with malagashanine and selected plant natural products

A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC₅₀ values of 8.5, 12, 50 and 69 μM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K_i for GSH and CDNB were ?0.015?μM and 0.011?μM, respectively. Malagashanine (100?µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t_1/2 of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5?mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds.     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity

Glutathione S-transferases (GSTs) are an important enzyme family which play a critical role in detoxification system. In our study, GST was purified from muscle tissue of Chalcalburnus tarichii Pallas with 301.5-fold purification and 19.07% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, showing a two band, because of having heterodimer structure. K_M values were 1.59 and 0.53?mM for 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), respectively. V_max values for CDNB and GSH were also determined as 5.58 and 1.88?EU/mL, respectively. In addition, inhibition effects of Ag⁺, Cu²⁺, Cd²⁺, Fe³⁺, Pb²⁺, Cr²⁺, Co²⁺ and Zn²⁺ metal ions were investigated on the enzyme activity and IC₅₀, K_i values were calculated for these metal ions.     

Hypochlorous acid inhibits glutathione S-conjugate export from human erythrocytes

It was found that the hypochlorous acid (HOCl) inhibits the active efflux of glutathione S-conjugates, 2,4-dinitrophenyl-S-glutathione (DNP-SG, c_50%=258±24 μM HOCl) and bimane-S-glutathione (B-SG, c_50%=125±16 μM HOCl) from human erythrocytes, oxidises intracellular reduced glutathione (the ratio [HOCl]/[GSH]_oxidized=4) and inhibits basal as well as 2,4-dinitrophenol- (DNP) and 2,4-dinitrophenyl-S-glutathione (DNP-SG)-stimulated Mg²⁺-ATPase activities of erythrocyte membranes. Multidrug resistance-associated protein (MRP1) mediates the active export of glutathione S-conjugates in mammalian cells, including human erythrocytes. A direct impairment of erythrocyte membrane MRP by hypochlorous acid was shown by electrophoresis and immunoblotting (c_50%=478±36 μM HOCl). The stoichiometry of the MRP/HOCl reaction was 1:1. These results demonstrate that MRP can be one of the cellular targets for the inflammatory mediator hypochlorous acid.     

Selective inhibition of glutathione S-transferases by 17 beta-estradiol disulfate.

Enzyme activity of homogeneous glutathione S-transferases A, B, and C with reduced glutathione and 1-chloro-2,4-dinitrobenzene was inhibited in varying degrees by 50 μm concentrations of monosulfate and disulfate derivatives of several steroids. In contrast, transferase AA activity was not affected. Of the inhibitors tested, estradiol-3,17-disulfate and estradiol-3-sulfate were the most inhibitory, followed by pregnenolone sulfate, estradiol-17-sulfate, dehydroisoandrosterone sulfate, and cortisol sulfate. Transferases A and C were most affected, especially by estradiol disulfate and estradiol-3-sulfate, which exhibited essentially complete inhibition at a concentration of μm. Double reciprocal plots of estradiol disulfate inhibition with respect to 1-chloro-2,4-dinitrobenzene concentration showed uncompetitive inhibition with transferases A and C and noncompetitive inhibition with transferase B (ligandin). With reduced glutathione as the variable substrate, transferases A and C exhibited noncompetitive inhibition kinetics, while transferase B showed partial noncompetitive kinetics.     

Purification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides

After cultivation on (R,S)‐2‐(2,4‐dichlorophenoxy)propionate, two α‐ketoglutarate‐dependent dioxygenases were isolated and purified from Delftia acidovorans MC1, catalysing the cleavage of the ether bond of various phenoxyalkanoate herbicides. One of these enzymes showed high specificity for the cleavage of the R‐enantiomer of substituted phenoxypropionate derivatives: the K_m values were 55 μM and 30 μM, the k_cat values 55 min^–1 and 34 min^–1 with (R)‐2‐(2,4‐dichlorophenoxy)propionate [(R)‐2,4‐DP] and (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. The other enzyme predominantly utilised the S‐enantiomers with K_m values of 49 μM and 22 μM, and k_cat values of 50 min^–1 and 46 min^–1 with (S)‐2‐(2,4‐dichlorophenoxy)propionate [(S)‐2,4‐DP] and (S)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4‐dichlorophenoxyacetate: K_m = 123 μM, k_cat = 36 min^–1) with significant activity. As the second substrate, only α‐ketoglutarate served as an oxygen acceptor for both enzymes. The enzymes were characterised by excess substrate inhibition kinetics with apparent K_i values of 3 mM with (R)‐2,4‐DP and 1.5 mM with (S)‐2,4‐DP. The reaction was strictly dependent on the presence of Fe²⁺ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 μM diethylpyrocarbonate (DEPC).     

Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver.

Glutathione $\text{S}$-transferases from rat lung cytosol were purified about 200-fold in one step by chromatography on $\text{S}$-hexylglutathione bound to epoxy-activated Sepharose 6B. Further purification on hydroxyapatite resolved the lung transferases into five peaks of activity as measured with 1-chloro-2,4-dinitrobenzene as substrate. Three of the peaks were identified with transferases A, B, and C of rat liver on the basis of chromatographic properties, immunochemical reactivity, and substrate specificity. The other two activity peaks were not detectable in liver: one originated from the lung tissue and one appeared to result from blood in the lung.     

Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K_M and V_max values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC₅₀ values, K_i constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag⁺; Cd²⁺, Cr²⁺ and Mg²⁺.     

Reduced glutathione esters—antidotes to toxicity. Cytotoxicity induced by hydrogen peroxide, 1-chloro-2,4-dinitrobenzene,and menadione in murine P388D1 macrophages in vitro

Repletion of depleted cellular reduced glutathione (GSH) levels in oxidative stress and exposure to arylating agents is a strategy for the development of antidotes to chemical toxicity. The effect of GSH, reduced glutathione ethyl monoester (GSHEt), and reduced glutathione ethyl diester (GSHEt₂) on the cytotoxicity of hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), and menadione to P388D₁ macrophages in vitro was investigated. The median toxic concentration TC₅₀ values of the toxicants were hydrogen peroxide 24 ± 2 mM (N = 19), CDNB 63 ± 6 μM (N = 18), and menadione 30 ± 4 μM (N = 22). Reduced glutathione, GSHEt, and GSHEt₂ were poor antidotes to hydrogen peroxide toxicity. Indeed, the observed antidote effects were attributed to the nonenzymatic reaction of the GSH derivatives with hydrogen peroxide in the extracellular medium. Reduced glutathione ethyl diester was a more potent antidote of CDNB- and menadione-mediated toxicity than GSHEt and GSH. For cell incubations with the approximate median toxic concentration TC₅₀ values of hydrogen peroxide, CDNB, and menadione, the respective median effective antidote concentration EC₅₀ values were GSHEt 23.8 ± 4.1 mM (N = 9), 3.6 ± 0.6 mM (N = 11), and 226 ± 93 μM (N = 12); and GSHEt₂ 20.4 ± 1.9 mM (N = 6), 603 ± 2 μM (N = 9), and 7.6 ± 2.3 μM (N = 12). Reduced glutathione ethyl diester was a potent antidote to CDNB- and menadione-induced toxicities but not to hydrogen peroxide-induced toxicity under acute intoxication conditions. © 1996 John Wiley & Sons, Inc.     

Comparison of glutathione S-transferases of Zea mays responsible for herbicide detoxification in plants and suspension-cultured cells

The metabolism of the s-triazine herbicide atrazine has been compared in Zea mays seedlings and cell suspension cultures. The rapid detoxification observed in the shoots of whole plants was not seen in the cultured cells. This difference in metabolism could be accounted for by the varying substrate specificities of the isoenzymes of glutathione S-transferase (EC 2.5.1.18) present in the plant and the cells. A single form of the enzyme isolated from leaf tissue conjugated both atrazine and the chloracetanilide herbicide metolachlor. However, the two isoenzymes present in suspension-cultured cells although active against metolachlor, showed no activity toward atrazine. Following purification, the major form of transferase present in the cells was physically similar to the enzyme isolated from leaf (M_r=55000). Both proteins were dimers of subunit M_r=26300, and with isoelectric points in the range pH 4.3-4.9. The minor form of the enzyme present in culture showed a greater specificity for metolachlor than the major species. In addition the overall activity and ratio of the two isoenzymes varied over the culture growth cycle. These findings illustrate the need for characterizing enzymes involved in herbicide detoxification in plant cell cultures.Abbreviations CDNB 1-chloro-2,4-dinitrobenzene - DEAE diethylaminoethyl - GSH glutathione (reduced) - GST glutathione S-transferase - HPLC high-pressure liquid chromatography - M_r molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Peroxide induced activation of glycogen phosphorylase A activity in vascular smooth muscle

1-Phenoxy-2-propanone, 1-chloro-3-phenoxy-2-propanone, and 1-fluoro-3-phenoxy-2-propanone are competitive acetylcholinesterase inhibitors with K_I values of 30, 0.85, and 2.2 μM, respectively, compared to 2 mM for 4-phenyl-2-butanone. The substituent effect on inhibition suggests that these compounds bind by formation of a tetrahedral adduct and are transition state analogs.Other evidence supports this conclusion: N-benzyl-2-chloroacetamide and 1-phenoxy-2-propanol are poor inhibitors (K_I = 11 and >10 mM); 1-phenoxy-2-propanone and 1-chloro-3-phenoxy-2-propanone have K_I values 330 and 140 times smaller than K_m for corresponding substrates; and 1-chloro-3-phenoxy-2-propanone protects the enzyme against irreversible inhibition by CH₃SO₂F.     

Acid phosphatases excreted by Humicola lutea 120-5 in casein-containing medium

The fungus Humicola lutea 120-5 cultivated in casein-containing media, in the presence or absence of inorganic phosphate (P_i), excretes three different molecular forms of acid phosphatase (with M_r values of approximately 140, 70 and 35 kDa). The enzyme forms were isolated and purified 30–100-fold by a procedure involving two steps of ion-exchange chromatography and Sephadex G-200 gel chromatography. It was found that the fungus excretes only one of the phosphatases with the highest M_r (140 kDa) during growth on medium with inorganic nitrogen source (NaNO₃). This form (designed AcPh I) was assumed to be a constitutive, since it showed resistance to high P_i-concentrations (10 mM) and its biosynthesis was not affected by the type of nitrogen source (casein or NaNO₃). The other two forms (AcPh II-70 and AcPh III-35 kDa) were competitively inhibited by P_i (K _i = 0.5 and 0.2 mM, respectively) and were induced by casein. The K _m values of AcPh I and AcPh II were estimated as 1.3 mM, while AcPh III showed a higher affinity for p-nitrophenylphosphate (pNPP) with K _m of 0.5 mM. The AcPh I–III fractions demonstrated a pH optimum in the range of 4.5–4.8 and an optimal temperature of 55 °C using pNPP as a substrate. This revised version was published online in November 2006 with corrections to the Cover Date.     

The interaction of cytosolic epoxide hydrolase with chiral epoxides

• 1.1. The kinetic parameters of the cytosolic epoxide hydrolase were examined with two sets of spectrophotometric substrates. The (2S,3S)- and (2R,3R)-enantiomers of 4-nitrophenyl trans-2,3-epoxy-3-phenylpropyl carbonate had a K_mof 33 and 68 μm and a V_max of 16 and 27 μmol/min/mg, respectively. With the (2S,3S)- and (2R,3R)- enantiomers of 4-nitrophenyl trans-2,3-epoxy-3-(4-nitrophenyl)propyl carbonate, cytosolic epoxide hydrolase had a K_M of 8.0 and 15 μM and a V_max of 7.8 and 5.0 μmol/min/mg, respectively.
• 2.2. Glycidyl 4-nitrobenzoate had the lowest I₅₀ of the compounds tested in the glycidyl 4-nitrobenzoate series (I₅₀= 140 μM). The I₅₀ of the (2R)-enantiomer was 3.7-fold higher. The inhibitor with the lowest i₅₀ in the glycidol series, and the lowest I₅₀ of any compound tested, was (2S,3S)-3-(4-nitrophenyl)glycidol (I₅₀ = 13.0μM). It also showed the greatest difference in I₅₀ between the enantiomers (330-fold).
• 3.3. All enantiomers of glycidyl 4-nitrobenzoates and _trans-3-phenylglycidols gave differential inhibition of cytosolic epoxide hydrolase. However, neither the (S,S)-/(S)- or (R,R)-/(R)-enantiomer always had the lower I₅₀.
• 4.4. Addition of one or more methyl groups to either enantiomer of glycidyl 4-nitrobenzoate resulted in increased I₅₀. However, addition of a methyl group to C₂ of either enantiomer of 3-phenylglycidol resulted in a decreased I₅₀. Finally, when the hydroxyl group of trans-3-(4-nitrophenyl)glycidol was esterified the I₅₀ of the (2S,3S)- but not the (2R,3R)-enantiomer increased.

     

Properties of larval and imaginal membrane-bound digestive enzymes from Trichosia pubescens

Trichosia pubescens larval midgut ceca cells display in their plasma membranes α-glucosidases (M_r 95,000; pH_o 5.5; K_m 5.7 mM; K_i for TRIS 8.9 mM), trehalases (M_r 69,000; pH_o 5.3; K_m 0.92 mM; K_i for TRIS 57 mM), and aminopeptidases (M_r 95,000; pH_o 8.7; K_m 0.19 mM) which are solubilized by Triton X-100. The enzymes were purified by electrophoresis and used to raise antibodies in a rabbit. T. pubescens imaginal midgut cells display in their plasma membranes an α-glucosidase (M_r 156,000; pH_o 5.8; K_m 2.3 mM; K_i for TRIS 0.2 mM), a trehalase (M_r 93,000; pH_o 5.5; K_m 0.72 mM; K_i for TRIS 45.5 mM), and an aminopeptidase (M_r 210,000; pH_o 9.0; K_m 0.47 mM). Antiserum produced against the larval enzymes shows no precipitation arc when tested by double immunodiffusion or by immunoelectrophoresis with Triton X-100-solubilized membrane proteins from imaginal midguts. Otherwise, a similar test showed that larval midgut cecal enzymes and larval ventriculus enzymes display complete immunological identity. The data suggest that, despite the fact the larval and imaginal aminopeptidase, α-glucosidase, and trehalase probably have similar functions, the genes coding for them in larvae and imagoes must differ.     

Purification and characterization of two glutathione peroxidases from embryo of the camel tick <Emphasis Type="Italic">Hyalomma dromedarii</Emphasis>

Two glutathione peroxidase isoenzymes were purified from 24-day old embryos of the camel tick Hyalomma dromedarii and designated tick embryo glutathione peroxidase 1 and 2 (TEGPx1 and TEGPx2). The purification procedure involved ammonium sulfate precipitation, as well as ion exchange and gel filtration column chromatography. Glutathione peroxidase isoenzymes subunit molecular mass was determined by SDS-PAGE to be 36 ± 2 kDa and 59 ± 1.5 kDa for TEGPx1 and TEGPx2, respectively. TEGPx1 isoenzyme exhibited a dimeric structure with native molecular mass of 72 kDa while TEGPx2 was a monomeric protein. TEGPx1 and TEGPx2 displayed their pH optima at 7.6 and 8.2. Both isoenzymes cleaved preferentially H₂O₂ with K _m values of 24 and 49 μM. Iodoacetamide competitively inhibited TEGPx1 with K _i value of 0.45 mM and 1.10; phenanthroline competitively inhibited TEGPx2 with K _i value of 0.12 mM. These results indicate the presence of two different forms of glutathione peroxidase in the developing camel tick embryos. This finding enhances our knowledge and understanding of the physiology of these ectoparasites and will encourage the development of new and untraditional control methods.