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Bruce D. Hammock - One of the best experts on this subject based on the ideXlab platform.
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structural studies of a potent insect maturation inhibitor bound to the Juvenile Hormone Esterase of manduca sexta
Biochemistry, 2006Co-Authors: Mark Wogulis, Bruce D. Hammock, Andrew C. Hinton, Craig E Wheelock, Shizuo G Kamita, Paul A Whetstone, David K WilsonAbstract:Juvenile Hormone (JH) is an insect Hormone containing an R,‚-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through Juvenile Hormone Esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent R/‚-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornwormManduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom ‚ to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.
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The Photorhabdus Pir toxins are similar to a developmentally regulated insect protein but show no Juvenile Hormone Esterase activity.
Fems Microbiology Letters, 2005Co-Authors: Nicholas R. Waterfield, Bruce D. Hammock, Shizuo G Kamita, Richard H. Ffrench-constantAbstract:The genome of the insect pathogen Photorhabdus luminescens strain TT01 contains numerous genes predicting toxins and proteases. Within the P. luminescens TT01 genome, the products of two loci, plu 4093-plu 4092 and plu 4437-plu 4436, show oral insecticidal activity against both moth and mosquito larvae. The proteins encoded by these loci, here termed ‘Photorhabdus insect related’ (Pir) proteins A and B, show similarity both to δ-endotoxins from Bacillus thuringiensis (Bts) and a developmentally regulated protein from a beetle, Leptinotarsa decemlineata. The beetle protein has been inferred to possess Juvenile Hormone Esterase (JHE) activity due to its developmentally regulated pattern of expression and the Photorhabdus proteins PirA and PirB have been proposed to be mimics of insect JHEs that can disrupt insect metamorphosis by metabolizing the insect growth regulator Juvenile Hormone (JH) [Nat. Biotechnol. 21 (2003) 1307–1313]. Here we confirm that, when injected together, PirA and PirB from two different Photorhabdus strains have insecticidal activity against caterpillars of the moth Galleria mellonella but show no oral activity against a second moth species Manduca sexta. Direct measurement of JHE activity, however, shows that the Pir proteins are not able to metabolise JH. These data show that the Pir proteins have no JHE activity, as suggested, but leave the mode of action of these interesting proteins uncertain.
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Expression of a Non-Secreted Form of Juvenile Hormone Esterase in a Baculovirus
Japan Agricultural Research Quarterly: JARQ, 2005Co-Authors: Kiyoko Taniai, Carol L. Ecale Zhou, Dennis Lee, Susumu Maeda, Bruce D. HammockAbstract:Pericardial cells rapidly cleared recombinant-Juvenile Hormone Esterase (rJHE) expressed within a baculovirus in insects from the hemolymph. To prevent the clearance of rJHE, we used the polymerase chain reaction (PCR) to remove the signal sequence from the JHE gene, thereby converting the enzyme to a non-secreted form (NSJHE). The resulting gene was expressed in a baculovirus (AcNSJHE) using HIGH-FIVE cells and proper cellular enzyme production was monitored. Transcription level of the NSJHE and rJHE were comparable, and purified NSJHE protein from the cytoplasm hydrolyzed JH. However, efficacy of NSJHE production was low. Enzyme-linked immunosorbent assays and enzyme assays demonstrated enzyme production and activity relative to rJHE of 0.5–1.7% and < 0.1%, respectively. Transmission electron microscopy (TEM) revealed that NSJHE was distributed in the nucleus predominantly and some NSJHE aggregated in clumps within the cytoplasm. These results indicate that NSJHE lacks a specific localization site within cells and that the folding of this enzyme is insufficient. Deglycosylation experiments using purified NSJHE showed that NSJHE was much less glycosylated than rJHE as we expected. Although the NSJHE was less glycosylated, enzyme stability of the NSJHE was equivalent to that of rJHE, indicating that the sugar chains are unimportant in the stability of JHE.
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use of classical and 3 d qsar to examine the hydration state of Juvenile Hormone Esterase inhibitors
Bioorganic & Medicinal Chemistry, 2003Co-Authors: Craig E Wheelock, Yoshiaki Nakagawa, Miki Akamatsu, Bruce D. HammockAbstract:CarboxylEsterases are important enzymes in the metabolism of numerous pharmaceuticals and agrochemicals. They are of importance in many detoxification pathways, but their endogenous role remains unclear. The most potent Esterase inhibitors found to date are trifluoromethylketone (TFK) containing compounds, which have been shown to inhibit both mammalian and insect Esterases at the low nM level. The detailed mechanism by which these compounds inhibit the enzyme is still unclear. They are highly hydrated in aqueous solutions, but their mechanism of inhibition suggests that inhibition occurs through the ketone, not the hydrated gem-diol. Some studies have stated that the ketone is the inhibitor, while others have reported the gem-diol as the active form. Using Juvenile Hormone Esterase (JHE) as a model system, we examined this question using both classical QSARand 3-D QSARwith comparative molecular field analysis (CoMFA). Classical QSARanalyses demonstrated the high dependence of inhi- bitor potency upon log P as well as the limitations of sterically unfavorable substituents. The ketone form of the inhibitor con- sistently provided improved correlations over the gem-diol, with the final equations describing 72 and 69% of inhibitor activity, respectively, for 97 compounds. Initial CoMFA analyses for the ketone provided a significant equation for 108 compounds (q 2 =0.412, m=6); however all cross-validated values for the gem-diol form of the inhibitors were not statistically significant (q 2 < 0.3). Inclusion of hydrophobicity descriptors in both CoMFA equations increased their significance; however the final ketone equation (q 2 =0.500, m=7) was still statistically improved over the gem-diol (q 2 =0.506, m=8). These results support those obtained for the classical QSARanalysis and further illustrate the importance of log P in the inhibition mechanism of these inhi- bitors. The CoMFA models also identified novel target areas for the synthesis of new JHE inhibitors. These results suggest that the ketone is the active form of TFK-containing inhibitors. # 2003 Elsevier Ltd. All rights reserved.
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Juvenile Hormone Esterase jhe from tenebrio molitor full length cdna sequence in vitro expression and characterization of the recombinant protein
Insect Biochemistry and Molecular Biology, 2003Co-Authors: Andrew C. Hinton, Bruce D. HammockAbstract:Juvenile Hormone regulates the development and reproduction in a variety of insects. Juvenile Hormone Esterase (JHE) is a selective enzyme, which hydrolyzes the methyl ester of JH and alters its activity. In Tenebrio molitor, JHE has been previously purified from pupae and a partial cDNA was amplified by RT–PCR using fat body mRNA. The previous report indicated that several forms of the JHE protein were present in pupal homogenate. In this study, we report the full-length cDNA, which was obtained by RACE methods. The deduced protein sequence corresponds to peptides from two proteins of different molecular weights in the previous study. The coding region of the full-length cDNA was subcloned into the AcMNPV genome and high levels of expression of the JHE enzyme from the viral p10 promoter were demonstrated in cell culture. The majority of JHE is secreted from the cells as a soluble enzyme. The recombinant JHE enzyme was biochemically characterized. The recombinant protein appears by PAGE analysis as a monomer of approximately the same MW (66,000) and pI (4.9) as was expected from the deduced amino acid sequence of the cDNA. 2003 Published by Elsevier Science Ltd.
Bryony C. Bonning - One of the best experts on this subject based on the ideXlab platform.
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potential ligands of dmp29 a putative Juvenile Hormone Esterase binding protein of drosophila melanogaster
Insect Biochemistry and Molecular Biology, 2007Co-Authors: Zhiyan Liu, Narinder Pal, Bryony C. BonningAbstract:We previously reported the identification of a putative Juvenile Hormone Esterase (JHE) binding protein DmP29 in Drosophila melanogaster and its primary localization to the mitochondria [Liu, Z., Ho, L., Bonning, B.C., 2007. Localization of a Drosophila melanogaster homolog of the putative Juvenile Hormone Esterase binding protein of Manduca sexta. Insect Biochem. Mol. Biol. 37(2), 155-163]. To further characterize DmP29, we identified potential ligands of this protein. Recombinant DmP29 was shown by ligand blot and co-immunoprecipitation analyses to bind recombinant JHE as well as to larval serum proteins (LSP). The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolyzed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant. Given the high abundance, and "sticky" nature of these proteins, interaction of DmP29 with LSP may result from non-specific associations. No DmP29 interactions with non-specific Esterases were detected by co-immunoprecipitation analyses. The potential role of DmP29 as a chaperone of JHE is discussed.
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localization of a drosophila melanogaster homolog of the putative Juvenile Hormone Esterase binding protein of manduca sexta
Insect Biochemistry and Molecular Biology, 2007Co-Authors: Zhiyan Liu, Bryony C. BonningAbstract:Abstract A putative Juvenile Hormone Esterase (JHE) binding protein, P29, was isolated from the tobacco hornworm Manduca sexta [J. Biol. Chem. 275(3), 1802–1806]. A homolog of P29 was identified in Drosophila melanogaster by sequence alignment. This gene, CG3776 was cloned, recombinant DmP29 expressed in Escheriscia coli and two anti-DmP29 antisera raised. In vitro binding of the P29 homolog to Drosophila JHE was confirmed. P29 mRNA and an immunoreactive protein of 25 kDa were detected in Drosophila larvae, pupae and adults. The predicted size of the protein is 30 kDa. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6 kDa N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25 kDa protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five TM cells showed that the N-terminal targeting sequence is shorter than predicted, and that a second, internal mitochondrial targeting signal is also present. An immunoreactive protein of 50 kDa in the hemolymph does not result from alternative splicing of CG3776 but may result from dimerization of P29. The function of P29 in mitochondria and the possible interaction with JHE are discussed.
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recombinant catalytically inactive Juvenile Hormone Esterase enhances efficacy of baculovirus insecticides
Biological Control, 2000Co-Authors: Marnix M M Van Meer, Bryony C. Bonning, Vernon K Ward, J M Vlak, Bruce D. HammockAbstract:Abstract The insecticidal efficacy of baculoviruses can be enhanced by engineering the viral genome to express proteins that disrupt the physiology of the host insect. Here we describe the development of a genetically engineered Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) which expresses a modified form of Juvenile Hormone Esterase (JHE). Previously, two viruses expressing different modified JHEs were found to have a greater insecticidal effect on larvae of Trichoplusia ni and Heliothis virescens than a virus expressing wild-type JHE. To study a possible synergistic effect, the distinct mutations in the modified JHEs were combined in a new JHE construct. Two lysine residues were replaced with arginine residues to reduce the efficiency of lysosomal targeting (JHE-KK) and the catalytic serine was replaced with glycine, which eliminated catalytic activity (JHE-SG). The modified JHE, JHE-KSK, was expressed in a recombinant baculovirus, AcJHE-KSK. Larvae of H. virescens infected with this recombinant virus caused 44% less feeding damage to lettuce than larvae infected with the wild-type AcMNPV. However, AcJHE-KSK did not have significantly improved insecticidal properties over the parent viruses AcJHE-KK and AcJHE-SG, suggesting that the separate mutations have no major synergistic effect. Infection with a control recombinant baculovirus expressing JHE with the same lysine to arginine conversions and in which a catalytic histidine was converted to lysine (AcJHE-KHK) did not reduce feeding damage compared with that caused by larvae infected with AcMNPV.
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a novel protein that binds Juvenile Hormone Esterase in fat body tissue and pericardial cells of the tobacco hornworm manduca sexta l
Journal of Biological Chemistry, 2000Co-Authors: Madasamy Shanmugavelu, Apollo R Baytan, Jonathan D Chesnut, Bryony C. BonningAbstract:Abstract Juvenile Hormone Esterase degrades Juvenile Hormone, which acts in conjunction with ecdysteroids to control gene expression in insects. Circulating Juvenile Hormone Esterase is removed from insect blood by pericardial cells and degraded in lysosomes. In experiments designed to characterize proteins involved in the degradation of Juvenile Hormone Esterase, a pericardial cell cDNA phage display library derived from the tobacco hornworm mothManduca sexta L. was constructed and screened for proteins that bind Juvenile Hormone Esterase. A 732-base pair cDNA encoding a novel 29-kDa protein (P29) was isolated. Western and Northern analyses indicated that P29 is present in both pericardial cell and fat body tissues and is expressed in each larval instar. In immunoprecipitation experiments, P29 bound injected recombinant Juvenile Hormone Esterase taken up by pericardial cells and nativeM. sexta Juvenile Hormone Esterase in fat body tissue, where the enzyme is synthesized. Binding assays showed that P29 bound Juvenile Hormone Esterase more strongly than it did a mutant form of the enzyme with mutations that perturb lysosomal targeting. Based on these data, we propose that P29 functions in pericardial cells to facilitate lysosomal degradation of Juvenile Hormone Esterase.
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Characterization and affinity purification of Juvenile Hormone Esterase from Bombyx mori.
Bioscience biotechnology and biochemistry, 2000Co-Authors: Takahiro Shiotsuki, Bryony C. Bonning, Makoto Hirai, Kyoko Kikuchi, Bruce D. HammockAbstract:Juvenile Hormone Esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the Juvenile Hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylEsterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine Esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.
Takahiro Shiotsuki - One of the best experts on this subject based on the ideXlab platform.
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depletion of Juvenile Hormone Esterase extends larval growth in bombyx mori
Insect Biochemistry and Molecular Biology, 2017Co-Authors: Takahiro Shiotsuki, Zhongjie Zhang, Xiaojing Liu, Zhisheng Wang, Yongping Huang, Anjiang TanAbstract:Two major Hormones, Juvenile Hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile Hormone Esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production.
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cdna cloning and characterization of bombyx mori Juvenile Hormone Esterase an inducible gene by the imidazole insect growth regulator kk 42
Insect Biochemistry and Molecular Biology, 2002Co-Authors: Makoto Hirai, Manabu Kamimura, K Kikuchi, Yuji Yasukochi, Makoto Kiuchi, Tetsuro Shinoda, Takahiro ShiotsukiAbstract:The insect growth regulator (IGR) imidazole KK-42 induces hemolymph Juvenile Hormone Esterase activity and precocious metamorphosis in Bombyx mori. As an initial step to understand the molecular action of KK-42, we isolated a full-length of Juvenile Hormone Esterase cDNA from B. mori (BmJHE). The deduced amino acid sequence of BmJHE showed high identity to JHEs of Heliothis virescens (54%) and Choristoneura fumiferana (52%). Recombinant BmJHE protein expressed in the baculovirus expression system hydrolyzed 3H-JH III and JH analog, HEPTAT, indicating that BmJHE cDNA encodes functional JH Esterase. Northern blot analysis showed that the BmJHE transcript was present predominantly in the fat body at the beginning of the last larval instar. During this instar, BmJHE transcript increased gradually until day 7, then decreased, and increased again on day 10 in the fat body. This temporary expression pattern was similar to that of JHE enzyme activity in hemolymph. In contrast, in the 4th instar, the BmJHE transcript was present in the fat body even though hemolymph JHE activity was very low. Western blot analysis using anti-BmJHE antiserum showed BmJHE protein was present in hemolymph during the 5th instar but not during the 4th instar. These results indicate that BmJHE protein is secreted into hemolymph at the metamorphic stage. Hemolymph JHE activity was high in precociously metamorphosed 4th instar larvae (treated KK-42) but low in normal 4th and extra-molted 6th instar larvae (fed 20E). KK-42-treated larvae showed high expression level of BmJHE transcript in the fat body, suggesting that KK-42 enhances BmJHE gene expression in the fat body.
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Fenoxycarb levels and their effects on general and Juvenile Hormone Esterase activity in the hemolymph of the silkworm, Bombyx mori
Pesticide Biochemistry and Physiology, 2002Co-Authors: Skarlatos G. Dedos, Takahiro Shiotsuki, András Székács, Jun Shimada, Hajime FugoAbstract:Abstract We studied the relationship between fenoxycarb levels and general Esterase (GE) activity, Juvenile Hormone Esterase (JHE) activity, and induction of permanent fifth instar (dauer) larvae in Bombyx mori . Fenoxycarb (1 μg/animal) was topically applied to B. mori at 0 h of the fifth instar. Hemolymph fenoxycarb levels, as determined by an ELISA, reached a peak within 1 h, and then decreased to undetectably low at 42 h. After 48 h, the feces of these larvae contained 99% of the applied fenoxycarb dose. The above fenoxycarb treatment induces dauer larvae with persistently increased hemolymph GE and JHE activities for the rest of the fifth instar. Application of fenoxycarb later in the fifth instar (48 h), when it still could induce dauer larvae, resulted in a similar pattern of elimination from the hemolymph and produced steadily elevated hemolymph GE and JHE activities for the rest of the fifth instar. Application of fenoxycarb late in the fifth instar (132 h) did not prevent pupal ecdysis; it increased hemolymph JHE activity, but not GE activity. The latter treatment prevented the JHE activity decrease observed in control larvae before pupal ecdysis. In vivo, in control animals, the highest hemolymph JHE activity was observed 12 h after pupal ecdysis. At that developmental time, fenoxycarb inhibited this enzyme in vitro ( IC 50 ≈2 μM ) . The combined results suggest: (1) fenoxycarb is rapidly excreted from the larval body; (2) induction of dauer larvae and increased hemolymph GE and JHE activities are not directly associated with its presence in the larval body of B. mori .
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Characterization and affinity purification of Juvenile Hormone Esterase from Bombyx mori.
Bioscience biotechnology and biochemistry, 2000Co-Authors: Takahiro Shiotsuki, Bryony C. Bonning, Makoto Hirai, Kyoko Kikuchi, Bruce D. HammockAbstract:Juvenile Hormone Esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the Juvenile Hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylEsterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine Esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.
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characterization of a spectrophotometric assay for Juvenile Hormone Esterase
Insect Biochemistry and Molecular Biology, 1995Co-Authors: Bill F Mccutchen, András Székács, Takahiro Shiotsuki, Tien L Huang, Bruce D. HammockAbstract:Abstract Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a new spectrophotometric assay with the standard radiochemical partition assay used to quantify Juvenile Hormone Esterase (JHE) activity. The surrogate substrates were made with one common factor being a thiol ester moiety substituting for the ester moiety found in Juvenile Hormones (JHs) and a thioether replacing the 2,3-olefin of the JHs. As a result, nucleophilic attack by the serine residue of JHE at the carbonyl functional group results in a hydrolytic reaction and release of methanethiol. In the presence of Ellman's Reagent (DTNB) methanethiol will cleave the disulfide bond of DTNB resulting in a chromophore detectable at 405 nm. Methyl 1-hexylthioacetothioate and its oxygen ester analogue, methyl-l-hexylthioacetate, were compared for JHE activity. Statistical analysis of the slopes indicated a very small but significant difference between the hydrolytic rates for the thiol ester and oxygen ester. However, the data indicate that thiol esters can replace oxygen esters to quantify hydrolytic activity by the JHEs examined. Results gathered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enzyme, as well as insect hemolymph indicate an excellent correlation between the two assays. Isoelectric focusing of pure and crude JHE preparations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either substrate. Several Esterase bands were found at different isoelectric points when gels were stained with a-naphthyl acetate. Substrates were further characterized by monitoring specific activity of JHE collected from hemolymph of larvae of Manduca sexta and Heliothis virescens
Anthony J. Zera - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of hemolymph Juvenile Hormone Esterase from the cricket gryllus assimilis
Archives of Insect Biochemistry and Physiology, 2002Co-Authors: Anthony J. Zera, Travis Sanger, Jeremiah Hanes, Lawrence G HarshmanAbstract:Juvenile Hormone Esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purifi ed to homogeneity in a four-step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900-fold purifi cation with greater than 30% recovery. The purifi ed enzyme exhibited a single band on a silver-stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryl- lus assimilis is a dimer of two identical or similar subunits. The turnover number of the purifi ed enzyme (1.41 s -1 ), K M(JH-III) (84 ± 12 nM) of nearly-purifi ed enzyme, and k cat /K M (1.67 × 10 7 s -1 M -1 ) were similar to values reported for other well-established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition-state analogue OTFP (octylthio-1,1,1-trifl uoro-2-propanone; I 50 = 10 -7 M) and by DFP (diisopropyl fl uorophosphate; I 50 = 10 -7 M). The shapes of the inhibition profi les suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Iso- electric focusing separated the purifi ed protein into 4 isoforms with pIs ranging from 4.7-4.9. N-terminal amino acid sequences (11-20 amino acids) of the isoforms differed from each other in 1-4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purifi ed JHE hydrolyzed α-napthyl esters, did not exhibit any detectable acetylcholines- terase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 μM) K M for long-chain α-naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH-III. Purifi cation of JHE represents a key step in our attempts to identify the molecular causes of genetically-based variation in JHE activity in G. assimilis. This represents the fi rst homogeneous purifi cation of JHE from a hemimetabolous insect.
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evolutionary endocrinology of Juvenile Hormone Esterase functional relationship with wing polymorphism in the cricket gryllus firmus
Evolution, 1999Co-Authors: Anthony J. Zera, Yuan HuangAbstract:The existence, nature, and physiological consequences of genetic variation for Juvenile Hormone Esterase (JHE) activity was studied in the wing-polymorphic cricket, Gryllus firmus. Hemolymph (blood) JHE activity was sixfold lower in nascent short-winged (SW) females, relative to nascent long-winged (LW) females during the last Juvenile stadium (stage). Morph-associated genetic variation for JHE activity had two causes, variation in loci: (1) regulating whole-organism enzyme activity; and (2) controlling the degree to which JHE is secreted into the blood Reduced JHE activity in nascent SW-selected individuals was associated with reduced in vivo Juvenile Hormone catabolism. This suggests that variation in JHE activity during Juvenile development may have important physiological consequences with respect to the regulation of blood levels of Juvenile Hormone and consequent specification of wing morph. This is the first definitive demonstration of genetic variation for hormonal metabolism in any insect and a genetic association between Hormone metabolism and the subsequent expression of morphological variation (wing morph). However, we have not yet firmly established whether these associations represent causal relationships In contrast to the clear association between JHE activity and wing morph development, we observed no evidence indicating that variation in JHE activity plays any direct or indirect role in causing the dramatic differences in ovarian growth between adult wing morphs. Variation in JHE activity also does not appear to be important in coordinating the development of wing morph with the subsequent expression of reproductive differences between adult morphs. Finally genetic variation for the developmental profiles of JHE activity during Juvenile and adult stages are remarkably similar in three Gryllus species. This suggests that genetic correlations between JHE activities during different periods of development, which underlie these activity profiles, have been conserved since the divergence of the three Gryllus species.
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Quantitative genetics of Juvenile Hormone Esterase, Juvenile Hormone binding and general Esterase activity in the cricket Gryllus assimilis
Heredity, 1996Co-Authors: Anthony J. ZeraAbstract:Quantitative genetics of Juvenile Hormone Esterase, Juvenile Hormone binding and general Esterase activity in the cricket Gryllus assimilis
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The role of Juvenile Hormone and Juvenile Hormone Esterase in wing morph determination in Modicogryllus confirmatus
Journal of Insect Physiology, 1996Co-Authors: Anthony J. Zera, Seiji TanakaAbstract:Abstract The role of Juvenile Hormone (JH) and Juvenile Hormone Esterase (JHE) in regulating wing morph determination was studied in the cricket Modicogryllus confirmatus . JHE activities were significantly higher in nascent long-winged (LW) vs short-winged (SW) crickets during the latter half but not during the first half of the last stadium. The magnitude and direction of the activity differences were similar to those previously documented between wing morphs of the cricket, Gryllus rubens . In contrast, activities of general Esterase, an enzyme or group of enzymes with no demonstrated role in regulating the JH titer in insects, showed no or only minor differences between morphs. The magnitude and direction of the JHE activity variation is consistent with a regulatory role for this enzyme in some aspect of wing dimorphism. However, the timing of the differences (exclusively during the last half of the last stadium) argue against a role in regulating wing length development per se . Single or multiple applications of Juvenile Hormone-III to nascent LW individuals during the first few days of the last stadium significantly redirected development from long to short wings. Multiple applications of acetone, by itself, also increased the production of short-winged adults. For most treatments, all individuals with shortened wings also had undeveloped flight muscles. These data suggest that JH may play a role in wing morph determination in M. confirmatus but that it affects a different aspect of the polymorphism from JHE.
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evolutionary endocrinology of Juvenile Hormone Esterase in gryllus assimilis direct and correlated responses to selection
Genetics, 1995Co-Authors: Anthony J. Zera, Caiqiu ZhangAbstract:Hemolymph Juvenile Hormone Esterase (JHE) activity on the third day of the last stadium in the cricket, Gryllus assimilis, exhibited a significant response to selection in each of six replicate lines. Mean realized heritability was 0.26 +/- 0.04. The response was due to changes in whole-organism enzyme activity as well as to changes in the proportion of enzyme allocated to the hemolymph compartment. In vivo Juvenile Hormone metabolism differed between some lines selected for high vs. low enzyme activity. Only minimal differences were observed between lines with respect to hemolymph protein concentration or whole-cricket activity of Juvenile Hormone epoxide hydrolase, the other major JH-degrading enzyme. Dramatic correlated responses to selection, equal in magnitude to the direct response, were observed for JHE activity on each of three other days of the last Juvenile stadium. In contrast, no correlated responses in JHE activity were observed in adults. This indicates that JHE activities throughout the last stadium will evolve as a highly correlated unit independent of adult activities and the evolution of endocrine mechanisms regulating Juvenile development can be decoupled from those controlling adult reproduction. This study represents the first quantitative-genetic analysis of naturally occurring endocrine variation in an insect species.
Tien L Huang - One of the best experts on this subject based on the ideXlab platform.
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characterization of a spectrophotometric assay for Juvenile Hormone Esterase
Insect Biochemistry and Molecular Biology, 1995Co-Authors: Bill F Mccutchen, András Székács, Takahiro Shiotsuki, Tien L Huang, Bruce D. HammockAbstract:Abstract Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a new spectrophotometric assay with the standard radiochemical partition assay used to quantify Juvenile Hormone Esterase (JHE) activity. The surrogate substrates were made with one common factor being a thiol ester moiety substituting for the ester moiety found in Juvenile Hormones (JHs) and a thioether replacing the 2,3-olefin of the JHs. As a result, nucleophilic attack by the serine residue of JHE at the carbonyl functional group results in a hydrolytic reaction and release of methanethiol. In the presence of Ellman's Reagent (DTNB) methanethiol will cleave the disulfide bond of DTNB resulting in a chromophore detectable at 405 nm. Methyl 1-hexylthioacetothioate and its oxygen ester analogue, methyl-l-hexylthioacetate, were compared for JHE activity. Statistical analysis of the slopes indicated a very small but significant difference between the hydrolytic rates for the thiol ester and oxygen ester. However, the data indicate that thiol esters can replace oxygen esters to quantify hydrolytic activity by the JHEs examined. Results gathered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enzyme, as well as insect hemolymph indicate an excellent correlation between the two assays. Isoelectric focusing of pure and crude JHE preparations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either substrate. Several Esterase bands were found at different isoelectric points when gels were stained with a-naphthyl acetate. Substrates were further characterized by monitoring specific activity of JHE collected from hemolymph of larvae of Manduca sexta and Heliothis virescens
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Juvenile Hormone Esterase purified by affinity chromatography with 8-mercapto-1,1,1-trifluoro-2-octanone as a rationally designed ligand.
Protein expression and purification, 1994Co-Authors: Takahiro Shiotsuki, Bryony C. Bonning, Tamon Uematsu, Tien L Huang, Vernon K Ward, Bruce D. HammockAbstract:Abstract Trifluoromethyl ketones are potent inhibitors of a variety of serine hydrolases. Based on this chemistry improved affinity chromatography procedures were developed for Juvenile Hormone Esterase from insects. New affinity gels were prepared by binding rationally designed ligands to epoxy-activated Sepharose. One ligand is 8-mercapto-1,1,1-trifluoro-2-octanone which has a methylene group replacing a sulfide sulfur β to the carbonyl of the trifluoromethyl ketone of the previously reported ligand, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-propanone. With many loading levels and Esterases, the original gel bound enzymes too tightly, resulting in elution difficulties. This replacement of the sulfur β to the ketone thought to interact with the catalytic serine decreases the binding capacity of the gel at similar loading by approximately 56% compared to the affinity gel with the thioether. However, elution of the enzyme from the column can be accomplished with less potent inhibitors such as 3- n -butylthio- or 3- n -pentylthio-1,1,1-trifluoro-2-propanone, which can easily be removed from the enzyme by dialysis in the presence of the detergent n -octyl β-D-glucopyranoside. An alternative approach allowing elution with less potent inhibitors involved varying concentrations of the previous high-affinity ligand to optimize the concentration of ligand on the column. Low concentrations of the high-affinity ligand also allowed the use of less potent eluting agents. These two improved affinity chromatography systems have been successfully used to purify Juvenile Hormone Esterase of Heliothis virescens to near homogeneity with a 30-90% recovery of recombinant Esterase secreted into the cell media in a baculovirus expression system. The purity of the Esterase after affinity chromatography with newly prepared gel was comparable to that produced using the original affinity system based on analyses by SDS-PAGE and isoelectric focusing. A library of affinity gels with ligands of different affinities used at several loading levels and a library of eluting inhibitors of varying potency facilitate the rational selection of conditions for the affinity purification of Esterases.
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development of surrogate substrates for Juvenile Hormone Esterase
Archives of Biochemistry and Biophysics, 1993Co-Authors: Bill F Mccutchen, Takahiro Shiotsuki, András Székács, Tamon Uematsu, Tien L Huang, Anne D Lucas, Bruce D. HammockAbstract:Twenty-nine thioester compounds were synthesized to test their effectiveness as surrogate substrates for the insect enzyme, Juvenile Hormone Esterase (JHE). Substrates were designed that resembled the endogenous substrate Juvenile Hormone (JH), with one common factor being a thioester instead of carboxyl ester found in JH. The principle of the spectrophotometric assay is based on a modification of Ellman's method. Characterization of the substrates showed that replacement of the carbon atom by a sulfur or oxygen beta to the carbonyl of the acyl group of the substrates resulted in an approximate five- to sixfold increase in the rate of hydrolysis by JHE. The specific activities of JHE, porcine liver carboxylEsterase, and acetylcholinEsterase were determined for the surrogate substrates. While JHE and porcine liver carboxylEsterase hydrolyzed several of the substrates, acetylcholinEsterase did not produce any detectable hydrolysis of the substrates. Michaelis-Menten kinetic parameters of the surrogate substrates when compared to a previously reported partition assay, utilizing radiolabeled [3H]JH III, indicated that the surrogate substrates have lower affinity as indicated by higher Km values but are more easily hydrolyzed (Vmax) by JHE. Furthermore, optimal reaction conditions for substrate hydrolysis and the spectrophotometric reaction were determined. In addition, first order rate constants for base hydrolysis and critical micelle concentrations were determined for several surrogate substrates. The spectrophotometric assay was also compared with a Vmax and research spectrophotometer, and these two instruments produced almost identical slopes. The relative potency of four transition state inhibitors of JHE was found to be similar with those of the surrogate substrates and the [3H]JH III substrate.
