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1.
Increased hydrolytic metabolism of organophosphate insecticides has been associated with resistance among Nebraska western corn rootworm populations. In this study, resistance-associated esterases were partially purified by differential centrifugation, ion exchange, and hydroxyapatite column chromatography, with a final purification factor of 100-fold and recovery of approximately 10%. Kinetic analysis of the partially purified enzyme indicated that the Km of the group II esterases was identical for the two populations, although Vmax was consistently threefold higher in the resistant population. A putative esterase, DvvII, was further purified to homogeneity by preparative polyacrylamide gel electrophoresis. DvvII is a monomer with a molecular weight of approximately 66 kDa, although three distinct isoforms with similar pIs were evident based on isoelectric focusing gel electrophoresis. Immunoassays with the Myzus persicae E4 antiserum indicated that group II esterases from D. v. virgifera were cross-reactive and expressed at much higher titers in the resistant population relative to the susceptible counterpart. These results suggest that the resistance is likely associated with overproduction of an esterase isozyme in resistant D. v. virgifera populations.  相似文献   

2.
A water-soluble Mg2+-dependent ATPase (coupling factor F1) was isolated from the mitochondria of housefly thorax. It comprised about 14% of the proteins from a crude preparation. The F1 preparation was nearly homogeneous as assessed by gel electrophoresis, isoelectric focusing, and electron microscopy. It was composed of five subunits with the following apparent molecular weights: α, 68,000; β, 61,000; γ, 38,000; δ, 27,000; and ?, 17,500. The isoelectric pH (pI) of this protein was 7.3. F1 had a pH optimum of 8.2 and a temperature optimum between 37 and 45°C. The enzyme was fairly stable at 25°C. Nearly complete loss of activity was noticed at 0°C, while at 0 or 25°C, glycerol (20%) partially stabilized the enzyme activity against such inactivation. The Km value of the enzyme with respect to ATP was 0.4 mM. The activity was stimulated by low concentrations of 2,4-dinitrophenol. The enzyme was inhibited by azide, p-hydroxymercuribenzoate, and guanidine hydrochloride. Oligomycin and the pesticides pyrethrin, cyhexatin, and DDT have no effect on the enzyme activity. However, all of these chemicals inhibited intact Mg2+- ATPase. The results are discussed in the light of differential responses of soluble and intact ATPase to these pesticides.  相似文献   

3.
Pyrethroid carboxyesterase which hydrolyzes the esters of chrysanthemumic acid was purified from rat liver microsome by cholic acid solubilization, ammonium sulfate fractionation, heat treatment, and DEAE-Sephadex A-50 column chromatography. The 45-fold purified enzyme (38% yield) is likely to consist of single protein, as evidenced by polyacrylamide gel disc electrophoresis and Sephadex G-100 column chromatography, and had a molecular weight of approximately 74,000 and a Km of 0.21 mM. It is susceptible to inhibition by organophosphates and carbamate insecticides and insensitive to pCMB, mercuric ion, and cupric ion. It is capable of hydrolyzing trans isomers of synthetic pyrethroids much more rapidly (five to ten times) than the cis counterparts. The purified pyrethroid carboxyesterase is apparently identical in nature with malathion carboxyesterase and with p-nitrophenyl acetate carboxyesterase.  相似文献   

4.
A “soluble” glutathione S-transferase that catalyzes the cleavage of the herbicide, 2,4′-dinitro-4-trifluoromethyl diphenylether (fluorodifen), was isolated and partially characterized from epicotyl tissues of pea seedlings. A 32-fold purification of the enzyme was achieved by differential centrifugation, ammonium sulfate precipitation, Sephadex gel filtration, and DEAE-cellulose ion exchange chromatography. The enzyme had a pH optimum of 9.3–9.5 and was specific for reduced glutathione, with an estimated apparent Km value of 7.4 × 10?4M. Limited specificity studies with four substituted 14C-labeled diphenylether compounds indicated that fluorodifen was the only effective substrate, with an estimated apparent Km value of 1.2 × 10?5M. Differences and similarities between the pea epicotyl enzyme and other plant and animal glutathione S-transferases were discussed from the standpoint of substrate specificity, pH optima, distribution, stability, and inhibitor studies.  相似文献   

5.
In the present study, we report on the changes observed in the kinetic properties of pyruvate kinase (PK) from whole body homogenate in the three developmental stages of the insect Tenebrio molitor after insecticide or acetone injection, and after injury and 24 h starvation. The apparent Km value of the enzyme towards phosphoenolpyruvate was higher in the pupae stage than in the rest developmental stages although the same enzyme type appears to be present throughout the life span of the insect as judged by its Vmax and sensitivity towards ATP and Alanine. The highest specific activity, twice as high as that of larvae and pupae, was observed in adults. Starvation resulted to activation of the enzyme by increasing the Km value in the two feeding stages, namely larvae and adult, while injury had the opposite effect. Acetone injection did not have any significant effect at any stage. Insecticide injection had varying effects, depending upon the developmental stage, the dose and the type of the insecticide. In larvae, low doses of methyl paraoxon and methyl parathion activated the enzyme but as the dose increased the Km value returned to normal levels. Low dose of decamethrin activated the enzyme also but at high dose it caused a severe inactivation as judged by a fivefold increase of the observed Km. In pupae, decamethrin had no effect, methyl parathion had similar effects to those observed in larvae, and methyl paraoxon caused activation of the enzyme at any dose given. In the adult stage none of the injected insecticides had any effect on the Km value although Vmax and specific activity were significantly altered.  相似文献   

6.
An insect chitin synthetase (CS) is readily assayed using the microsomal fraction (～0.5 mg protein) from an homogenate of Tribolium castaneum larvae. This enzyme preparation is incubated at 22°C with uridine 5′-diphospho-N-acetyl[3H]glucosamine in 355 μl of 25 mM Tris-HCl buffer containing 10 mM MgCl2, 17 mM N-acetylglucosamine, and 1 mM dithiothreitol. Other divalent cations and amino sugars are less effective activators or are inhibitory. T. castaneum CS is strongly inhibited by polyoxin D and uridine 5′-diphosphate. These activation and inhibition properties of Tribolium castaneum gut CS are similar to those of fungal CS. The polymerization product formed by the Tribolium enzyme is stable in alkali but hydrolyzed by chitinase. Enzymes of Tribolium confusum, Tribolium brevicornis, Tenebrio molitor, and Galleria mellonella are also active under the same conditions. These enzymes are from the gut and probably from the peritrophic membrane. Integumental CS activity is not detected under the indicated assay conditions.  相似文献   

7.
l-[U-14C]sucrose accumulation by phloem sieve tube members (PSTM) of wheat (Triticum aestivum L. ‘Holley’) and sorghum (Sorghum bicolor L. ‘G522 DR’) was inhibited by the nonpermeant sulfhydryl inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), and this inhibition was reversed by the permeant sulfhydryl protectants dithiothreitol (DTT) and dithioerythritol (DTE). S-Ethyl dipropylthiocarbamate (EPTC) (≤0.1 mM) did not inhibit [14C]sucrose accumulation by wheat or sorghum PSTM. N-N-Diallyl-2-chloroacetamide (CDAA) (1 mM) inhibited [14C]sucrose accumulation by sorghum but not by wheat PSTM. The inhibition of [14C]sucrose accumulation in sorghum PSTM by the membrane permeant CDAA was reversed by DTT. Sorghum growth was inhibited by <1 μM CDAA. Membrane permeant 2-chloroallyl diethyldithiocarbamate (CDEC) (0.1 mM) inhibited [14C]sucrose accumulation by PSTM of sorghum but not wheat. The inhibition of sucrose accumulation in sorghum PSTM by 0.1 mM CDEC was reversed by DDT.  相似文献   

8.
An esterase which hydrolyzes the ester bond of trans-permethrin has been partially purified from homogenates of larvae of the cattle tick. The final (gel filtration) step showed two distinct peaks of p-nitrophenylbutyrate-hydrolyzing activity. The trans-permethrin-hydrolyzing activity of the lower-molecular-weight enzyme cochromatographed with the p-nitrophenylbutyrate-hydrolyzing activity of that enzyme. Little trans-permethrin hydrolysis was observed in the high-molecular-weight peak. The yield of the low-molecular-weight enzyme increased on extraction of the homogenates with Triton X-100. Inhibition studies using the low-molecular-weight enzyme and trans-permethrin as substrate indicated that hydrolysis was due largely to a carboxylesterase (EC 3.1.1.1).  相似文献   

9.
An enzyme that possesses the glutathione S-transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS-polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3-10 and at temperatures below 50 °C. The enzyme was also able to conjugate glutathione to 4-hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.  相似文献   

10.
Midgut juice of Plutellaxylostella strain PXR which is resistant to Cry1Ac was biochemically characterized relative to the susceptible PXS strain. The midgut juice of PXR (PXR-Juice) was shown to process Cry1Ac protoxin to 60 kDa active toxin with the same processing pattern as that of juice from PXS (PXS-Juice) in SDS–PAGE. PXS larvae which were given the Cry1Ac toxin pre-processed with PXR-Juice were killed with the same rate as that with Cry1Ac pre-activated by trypsin. PXR-Juice was found to contain three times larger amount of 66 kDa protein (P66) than PXS-Juice and the N-terminal amino acid sequence of P66 was matched to that of glucosinolate sulfatase in data base search. The protein band of P66 was coincided with the band of p-nitro phenyl sulfatase activity in zymogram. P66 purified to homogeneity in SDS-PAGE bound to Cry1Ac and soybean agglutinin, and KD for Cry1Ac was estimated to be 718 nM with surface plasmon resonance analysis. Using purified sulfatase, Km and Vmax were estimated and involvement of the enzyme in the PXR resistance was discussed.  相似文献   

11.
The structural gene for glutathione S-transferase in Oryza sativa was successfully cloned from a cDNA library by the polymerase chain reaction method. The deduced amino acid sequence of this gene showed 44-66% similarity to the sequences of the class phi GSTs from Arabidopsis thaliana and Zea mays. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF3-3 was a homo-dimer composed of 24 kDa subunit and its pI value was approximately 7.3. The OsGSTF3-3 was retained on GSH affinity column and its Km value for GSH was 0.28 mM. The OsGSTF3-3 displayed high activity toward 1-chloro-2,4-dinitrobenzene, a general GST substrate and also had high activities towards acetanilide herbicides, alachlor, and metolachlor. The OsGSTF3-3 was highly sensitive to inhibition by benastatin A and S-hexyl-GSH. From these results, the expressed OsGSTF3-3 is a phi class GST and seems to play an important role in the conjugation of the chloroacetanilide herbicides.  相似文献   

12.
Properties of acetolactate synthase (EC 4.1.3.18; ALS) from sulfonylurea-resistant (SUR) Scirpus juncoides Roxb. var. ohwianus T. Koyama were studied biochemically and physiologically in comparison with those from sulfonylurea-susceptible weed (SUS). GR50 values for growth inhibition and I50 values for ALS inhibition by imazosulfuron were determined for both SUR and SUS. Imazosulfuron controlled the SUS above 80% at the dosage more than 10 g a.i./ha but did not control the SUR at the even great dosage of 1000 g a.i./ha. The rates required for 50% growth inhibition of the SUR relative to the SUS (R/S ratio) were 271-fold. The I50 value for inhibition of ALS from the SUS was 15 nM, compared to I50 of >3000 nM for inhibition of ALS from the SUR. These results suggest that a resistance may due to an altered ALS that is insensitive to imazosulfuron. The Km (pyruvate) value of ALS from the SUR was similar to the Km for ALS from the SUS, suggesting that a mutation resulting in resistance does not change the affinity of the enzyme for pyruvate. The specific activity of the SUR ALS was similar to that of the SUS ALS, which indicates that resistance is not an over-expression of the enzyme. ALS activity from both biotypes was inhibited by isoleucine, valine, and leucine in this order. However, the SUR ALS was less sensitive to inhibition by valine than the SUS ALS.  相似文献   

13.
Malathion resistance in Anopheles arabiensis from Sudan is monofactorially inherited and is expressed in the adults but not in the larvae. The resistance is suppressed by the esterase synergist, triphenylphosphate. Semipurification of the soluble esterase enzymes by Sephadex G-25 and Sephacryl S-200 gel filtration revealed no difference between the enzymes of the resistant and susceptible strains with α- or β-naphthylacetate (NA) with a fixed substrate concentration in either the adults or larvae. However, with the malathion-specific assay a second peak of activity was observed in the adult resistant strain which was not present in either the larvae of this strain or the larvae and adults of the susceptible strain. A corresponding threefold difference in the Km value for α-NA was also observed in the resistant adults over the range of this second peak, but there was no change in the Km with β-NA.  相似文献   

14.
Chitin polymerization is catalyzed by cell-free enzyme complexes from the integument of Trichoplusia ni larvae and wing tissue of developing Hyalophora cecropia pupae obtained on extraction of homogenates for 16 hr at 5°C in 25 mM Tris-HCl buffer, pH 7.2, containing 10 mM MgCl2, 1 mM dithiothreitol, 10 mg/ml bovine serum albumin, and 4 mg/ml digitonin. In contrast, integumental preparations from Boarmia selenaria, Earias insulana, Heliothis virescens, Oncopeltus fasciatus, Spodoptera exigua, and Tribolium castaneum exhibit little or no chitin synthetase (CS) activity. H. cecropia CS requires magnesium ions but not N-acetyl-d-glucosamine for normal activity and is almost insensitive to nikkomycin and polyoxin D. CS activity is not detected in diapausing H. cecropia pupae but synthesis of this enzyme is induced or its activity is stimulated by the molting hormone, ecdysterone, indicating possible hormonal control. T. ni CS requires magnesium ions and N-acetyl-d-glucosamine for optimal activity and is sensitive to inhibition by uridine di- and triphosphates, polyoxins B and D, and particularly nikkomycin. T. ni integumental CS activity decreases in starved larvae or those about to pupate. Both T. ni and H. cecropia CS enzymes as prepared and assayed are sensitive to captan but not to the potent insecticides diflubenzuron and BAY SIR 8514, two effective benzoylphenyl urea in vivo chitin synthesis inhibitors.  相似文献   

15.
The phytotoxicities of nine pesticides (paraquat, fluazifop-p-butyl, haloxyfop, flusilazole, cuproxat, cyazofamid, imidacloprid, chlorpyrifos, and abamectin) at practical dosages on photosynthesis were investigated in cucumber (Cucumis sativus L. cv. Jinyan No. 4) by gas exchange and chlorophyll fluorescent measurements. Plants treated with paraquat showed the severest phytotoxic symptom with the highest reduction in net photosynthetic rate (Pn), while other pesticides except flusilazole inhibited Pn to various degrees. The inhibition of Pn by cuproxat was accompanied by declines both in stomatal conductance (Gs) and intercellular CO2 concentration (Ci), whereas decreased Pn for the cyazofamid was associated with increased Ci. For other 6 pesticides, however, inhibition of Pn was accompanied by decrease in Gs, while Ci was increased or unaffected. Paraquat almost completely inhibited the maximal quantum efficiency of PSII (Fv/Fm), while other pesticides had no significant effect on Fv/Fm. Quantum efficiency of PSII (ΦPSII) was significantly reduced by paraquat, fluazifop-p-butyl, and chlorpyrifos and the reduction was mostly attributed to decrease in photochemical quenching coefficient (qP). In comparison, ΦPSII was not significantly affected by haloxyfop, flusilazole, cyazofamid, imidacloprid, and abamectin. Non-photochemical quenching (NPQ) was suppressed by paraquat and haloxyfop, while apparent upregulation was evident after exposure to other pesticides. Interestedly, inhibitions of Pn were alleviated by 24-epibrassinolide (EBR) pretreatment, as for the pesticides examined in this study except paraquat and flusilazole. EBR pretreatment also increased ΦPSII and qP. It is likely that EBR enhanced the resistance of cucumber seedlings to pesticides by increasing CO2 assimilation capacity and activities of antioxidant enzymes.  相似文献   

16.
Four major esterases in one susceptible (CSMA) and two resistant (Hirokawa, E1) house fly strains were separated by chromatofocusing. Of the four esterases, those with pI's of 5.1 and 5.3 accounted for 90% of the p-nitrophenyl butyrate hydrolyzing activity in the three house fly strains. They also accounted for 70% (Hirokawa, E1) and 40% (CSMA) of the paraoxon-hydrolyzing activity as well as 87% (Hirokawa), 39% (E1) and 66% (CSMA) of the malathion-hydrolyzing activity in microsomes as measured by esterase-antibody interaction. In the Hirokawa strain, the pI 5.1 esterase was the predominant esterase and was more active than that of the the CSMA strain. Different substrate specificities and a different Km toward acetylthiocholine, as well as different rates of malathion and paraoxon hydrolysis between the Hirokawa and CSMA strains, suggest a qualitative difference in the pI 5.1 esterase. For the pI 5.1 esterase from the E1 strain, a different substrate specificity, a different Km for p-nitrophenyl butyrate, a different sensitivity to inhibitors, and a different rate of paraoxon hydrolysis suggest that it is a modified esterase. This esterase is not a phosphorotriester hydrolase, nor does it lack nonspecific esterase activity. It is a modified esterase which has a different substrate specificity when compared to the esterases from the other strains. The molecular weight of the esterases studied was approximately 220,000, with pH optima of about 7.0.The ratio of malathion α-monoacid to β-monoacid formation was about 9.0 for the pI 5.1 and 5.3 esterases and 1.5 for the pI 4.8 and 5.6 esterases. The existence of a higher αβ ratio for the pI 5.1 and 5.3 esterases and their significant rate of malathion hydrolysis in the Hirokawa strain indicate that an increase in the αβ ratio in house flies reported was due to the increase in the pI 5.1 esterase in the resistant strain.  相似文献   

17.
Four soluble acetylcholinesterase isozymes of head and three from the thorax of housefly (Musca domestica L.) were separated by polyacrylamide gel electrophoresis. Their inhibition in vivo was studied after poisoning with an LD50 of four organophosphates: malaoxon, paraoxon, diazinon and dichlorvos. The isozymes differed greatly in their degree of inhibition. Thoracic isozymes were found to be more sensitive to inhibition than head isozymes. In surviving flies, the recovery rates of head isozymes were much faster than thoracic isozymes. In vitro studies showed that thoracic isozymes differed 4.1- and 2.9-fold in their Km and Vmax.  相似文献   

18.
The ability of purified microsomal FAD-containing monooxygenase from mouse and pig liver to oxidize pesticides has been investigated. The kinetic constants, Km and Vmax, were determined for a number of pesticide substrates including thioether-containing organophosphorus compounds and carbamates as well as (di)alkyldithiocarbamates. In general, the mouse liver enzyme had Km values higher than those of the pig liver enzyme. Values for Vmax were similar regardless of substrate, although the Vmax typical of the mouse liver enzyme was approximately twice that of the pig liver enzyme. The thioether-containing organophosphorus compounds were the best substrates for both enzymes followed by the thioether-containing carbamates. The (di)alkyldithiocarbamates were relatively poor substrates for both pig and mouse liver microsomal FAD-containing monooxygenases.  相似文献   

19.
Five acetylcholinesterase isozymes from electric eel eletroplax (Electrophorus electricus) and four from housefly brain (Musca domestica L.), were separated by polyacrylamide gel electrophoresis combined with specific substrate staining and gel scanning procedures. The Km values for acetylthiocholine varied over a 2.4-fold range for eel and a 4.2-fold range for housefly. The sensitivities to inhibition also varied: for eel, the range was 2.2-fold for malaoxon, 2.6-fold for Tetram and 2.1-fold for eserine. The corresponding values for housefly were 2.3, 1.5, and 1.9.  相似文献   

20.
Kinetic parameters were measured for glutathione S-transferase, an enzyme important in metabolic resistance to insecticides, in one susceptible and two insecticide-resistant strains of the house fly (Musca domestica L.), and in untreated and chemically induced flies. Both resistant strains differed from the susceptible strain in apparent Km values for the enzyme, while only one differed in apparent Vmax. Two of the strains were inducible with phenobarbital; the third with 3-methylcholanthrene. Kinetic analysis indicated enzyme induction was associated with changes in Km rather than Vmax, and genetic experiments showed that most variation relating to Km and Vmax was controlled by chromosome II. Based on these results, both metabolic resistance and induction of enzyme activity were associated primarily with the production of different forms of glutathione S-transferase rather than more of the enzyme present in susceptible flies.  相似文献   

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