Purification, Biochemical And Toxicological Characterization Of Glutathione S-transferases From Psocids

Psocids,belonging to Liposcelis(Psocoptera:Liposcelididae),are worldwide and commonly found in various processed and unprocessed dry foods.To date there are 123 described species in total and 27 species have been recorded in China.Outbreaks of L.bostrychophila,together with L. entoraophila could pose an alarming threat to stored product and bring about serous economic losses.In addition,the rapid development of resistance to chemical and physical treatments by the psocids has also been widely reported.Separation and purification is the basis for protein structure and function study.The separation of a specific enzyme via protein purification methods,and the acknowledgment of the relationship between their biochemical properties and resistance to insecticides possess important significance in pest insect management.Glutathione S-transferases(GSTs,EC 2.5.1.18) are a supergene family of multifunctional enzymes found ubiquitously in mammals,insects,plants and aerobic bacteria.GSTs can metabolize foreign compounds by facilitating their reductive dehydrochlorination or by conjugation reactions with reduced glutathione,to produce water-soluble metabolites that are more readily excreted.In addition,they are involved in intracellular transport and biosynthesis of hormones,removal of toxic oxygen free radicals produced through the action of xenobiotics and protection against oxidative stress.On the basis of GSTs purification of L.bostrychophila and L. entomophila through the affinity chromatography method,the biochemical and toxicological characterizations were systematically analyzed.The study results provide biochemical and toxicological evidence for clarifying the adaptive mechanism of psocids to environment,and meanwhile enrich and develop the scientific theoretical study system for the resistance mechanism of stored product pests.The project is supported by the Program for New Century Excellent Talents in University(NCET-04-0854) and the Specialized Research Fund for the Doctoral Program of Higher Education(200806350009),and the main results are as follows:1 Purification,biochemical and toxicological characterization of GSTs from three strains of L.bostrychophilaGSTs from three strains of L.bostrychophila(SS:susceptible strain;DDVP-R,dichlorvos-resistant strain;PH₃-R,phosphine-resistant strain) were purified through Glutathione Sepharose 4B affinity chromatography method and the purified product amounted to electrophoresis-grade purity. SDS-PAGE revealed that the molecular mass of purified GSTs was estimated at approximately 23 kDa.The N-terminus of the purified enzyme was sequenced by Edman degradation method,and a primary structure of APKYKYL TYPFNSILTGLGL was obtained.The alignment of N-terminal sequence with other insect GSTs suggested the purified protein was similar to those of Sigma class GSTs.Purification fold of GSTs from three strains of L.bostrychophila ranged from 62 to 91 with a yield rate from 27%to 41%.The biochemical characterization result revealed that compared to SS strain,the specific activities of GSTs in DDVP-R and PH₃-R were significantly higher(P＜0.05) but no significance observed between two resistant strains(P＞0.05).Further kinetic parameters comparison showed that GSTs from DDVP-R expressed highest K_m value to the substrate CDNB while both two resistant strains exhibited lower K_m values to GSH.For V_max,the values in resistant strains were significantly higher suggesting the catalytic ability of GSTs developed to higher levels in two resistant strains.The pH optimum study showed that GSTs of SS strain possessed a wider optimal pH range(pH 7.0 to pH 8.0) and the optimal pH for resistant strains lied in pH 7.0 to pH 7.5.Temperature optimum of enzymatic reaction revealed that GSTs from SS and PH₃-R strains possessed highest specific activities in 35℃while activity peak recorded at 40℃for GSTs of DDVP-R strain.In vitro inhibition of insecticides showed that carbosulfan expressed significant inhibitory effects on GSTs in three strains of L.bostrychophila and the sensitivity of GSTs from PH₃-R was lowest.For beta-cypermethrin and chlorpyrifos,the inhibition effects were not obvious and some facilitated effects appeared at lower concentrations of chlorpyrifos.Ethacrynic acid,curcumin and bromosulfalein all exhibited significant in vitro inhibition effects.Among these inhibitors,GSTs were most sensitive to curcumin with its I₅₀ values at nanomolar concentrations.The comparison analysis revealed that compared to the counterparts in resistant strains GSTs from SS strain were most sensitive and sensitivity difference to inhibitors existed for GSTs in resistant strains.2 Purification,biochemical and toxicological characterization of GSTs from different developmental stages of L.bostrychophilaGSTs from different developmental stages of L.bostrychophila(Early nymph:1st and 2nd nymph;Late nymph:3rd and 4th nymph;Adult) were purified through Glutathione Sepharose 4B affinity chromatography method.The result revealed that the purification fold of GSTs from early nymph was highest(102) with a low yield rate of 28%,while late nymph exhibited lowest purification fold(63) with a yield rate of 61%.The specific activity comparison showed that highest value observed in adults.Further kinetic parameters comparison found GSTs from early nymph expressed lowest K_m values to the substrates CDNB and GSH while adults possessed highest V_max values,suggesting GSTs with higher catalytic ability to the substrates existed in adults. In vitro inhibition of chlorpyrifos showed that the pesticide expressed some inhibitory effects. At lower assay concentrations of chlorpyrifos some facilitated effects occurred while at higher concentrations inhibition appeared.The highest inhibition rate reached 39%under the test concentrations of chlorpyrifos.All five chemicals(ethacrynic acid,curcumin,disulfiram,diethyl maleate and bromosulfalein) exhibited significant in vitro inhibition on GSTs from different developmental stages of L.bostrychophila.Among these inhibitors,GSTs were least sensitive to disulfiram with its I₅₀ values at millimolar concentrations.The comparison analysis revealed that GSTs in adults were most tolerant to all other inhibitors except that GSTs in late nymph least sensitive to ethacrynic acid.Besides,the effects of metal ions(Zn²⁺,Hg²⁺,Mn²⁺,Cu²⁺ and Ca²⁺) on GSTs of different stages of L.bostrychophila were assayed.The results suggested that all metal ions expressed significant inhibitory effects.Among these metal ions,Ca²⁺ exhibited least insensitivity with its I₅₀ value at nanomolar levels.The effects of Hg²⁺ were most significant but no significant difference among different stages of L.bostrychophila(P＞0.05).3 Purification,biochemical and toxicological characterization of GSTs from different populations of L.bostrychophilaGSTs from different populations of L.bostrychophila(Guanghan population:from Guanghan of Sichuan province;Jianyang population:from Jiangyang of Sichuan province;Lab conservation population) were purified through Glutathione Sepharose 4B affinity chromatography method.The result revealed that the purification fold of GSTs from Lab population was highest(121) with a highest yield rate of 52%,while Jianyang population exhibited lowest purification fold(40) with a lowest yield rate of 16%.The purification parameters of Guanghan population ranged between those of the other populations.Both the total protein and specific activity comparison showed that highest values recorded in Lab population but no significance occurred between the parameters of other two populations.Further kinetic parameters comparison found GSTs of Lab population expressed lowest affinity ability to the substrates CDNB and GSH,Jianyang population possessed highest affinity ability to GSH and Guanghan population showed highest affinity ability to CDNB.For V_max values, Lab population revealed highest catalytic activity to both substrates,significantly higher than Jianyang and Guanghan populations(P＜0.05).In vitro inhibition of insecticides on GSTs of different populations of L.bostrychophila showed that Guanghan population was most sensitive to carbosulfan and Lab population possessed least sensitivity to the insecticide.For beta-cypermethrin,the inhibition effects were not obvious and the situation in different populations was similar.Ethacrynic acid,curcumin and diethyl maleate all exhibited significant in vitro inhibition.Among these populations,Guanghan population expressed most sensitivity to all inhibitors.Besides,the effects of metal ions(Zn²⁺,Hg²⁺,Mn²⁺,Cu²⁺ and Ca²⁺) on GSTs of different populations of L.bostrychophila were assayed.The results suggested that all metal ions expressed significant inhibitory effects.Among these metal ions,Ca²⁺ exhibited least insensitivity with no significant difference among different populations(P＞0.05).Similar situation observed for Mn²⁺ and Cu²⁺,Guanghan population was most sensitive to these ions while Lab population was least sensitive.4 Purification,biochemical and toxicological characterization of GSTs from different populations of L.entomophilaGSTs from different populations of L.entomophiIa(Kaifeng population:from Kaifeng of Henan province;Guanghan population:from Guanghan of Sichuan province;Lab population:from Beibei of Chongqing city;Wuhan population:from Wuhan of Hubei province) were purified through Glutathione Sepharose 4B affinity chromatography method.The result revealed that the purification fold of GSTs from Kaifeng population was highest(162) with a yield rate of 29%,Guanghan population exhibited lowest purification fold(57) with a yield rate of 43%,Lab population possessed highest yield rate of 47%with a purification fold of 93 and the lowest yield rate was observed in Wuhan population.The activity assay showed that Kaifeng and Wuhan populations expressed relatively higher catalytic activity(P＞0.05),no significant difference between these two populations but Kaifeng population exhibited lowest affinity ability to the substrates GSH and CDNB.The pH optimum study showed that the optimal pH for GSTs of Guanghan population of L. entomophila ranged from pH 6.5 to pH 7.0 while the range for other three populations lies in pH 7.0 to pH 7.5.Temperature optimum of enzymatic reaction revealed that GSTs from Lab population possessed highest specific activity in 40℃while activity peak recorded at 35℃for GSTs of other populations.Ethacrynic acid,curcumin,diethyl maleate and bromosulfalein all exhibited significant in vitro inhibition effects.The comparison analysis revealed that GSTs of Kaifeng population were least sensitive to all test inhibitors,Guanghan population exhibited most sensitivity to ethacrynic acid and diethyl maleate while Lab population expressed most sensitivity to curcumin and bromosulfalein.