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 (66000) and pI (4.9) as was expected from the deduced amino acid sequence of the cDNA.
        
Title: In vitro expression and biochemical characterization of juvenile hormone esterase from Manduca sexta Hinton AC, Hammock BD Ref: Insect Biochemistry & Molecular Biology, 33:317, 2003 : PubMed
Juvenile hormone esterase (JHE) is a selective enzyme that hydrolyzes the methyl ester of juvenile hormone. This enzyme plays an important role in the regulation of metamorphosis in caterpillars, and is implicated in additional roles in development and reproduction in this and other orders of insect. The full length coding region of the JHE cDNA from Manduca sexta was subcloned into the baculovirus AcMNPV genome under the control of the p10 promoter. The recombinant virus demonstrated the expression of high levels of JHE activity when infected into Hi5 cells from Trichoplusia ni. The recombinant protein was partially purified by anion exchange chromatography and its biochemical characterization showed similar features to the wild type protein. The recombinant JHE has an estimated MW of 66500 Da. Some heterogeneity with the enzyme was observed when analyzed by isoelectric focusing, although the peak of JHE activity was observed at pI=6.0. It is highly sensitive to trifluoroketone inhibitors and certain phosphoramidothiolates, while relatively insensitive to other common esterase inhibitors. Incubating the enzyme with various organic solvents and detergents showed that the enzyme is activated at lower concentrations of solvents/detergents and remains significantly active even at high concentrations. The high tolerance of organic solvents may make this JHE enzyme useful in future applications as a synthetic catalyst.
        
Title: Insecticidal, anticholinesterase, and hydrolytic properties of S-aryl phosphoramidothioates Sanborn JR, Fukuto TR Ref: Journal of Agricultural and Food Chemistry, 20:926, 1972 : PubMed
The insecticidal, anticholinesterase, and hydrolytic properties for a series of S-phenyl phosphoramidothioates and 5-phenyl phosphonamidothioates were examined. The compounds were moderately toxic to the housefly and were effective inhibitors of cholinesterase. Attempts to correlate cholinesterase inhibition of housefly toxicity with physical organic parameters were unsuccessful. However, an excellent linear relationship was obtained between Hammett's sigma constant and alkaline hydrolysis rates of the O-ethyl substituted S-phenyl phosphoramidothioates. In addition, a kinetic study of the alkaline hydrolysis of these esters was carried out for the purpose of examining the mechanism of reaction. The results indicate that hydrolysis takes place by a direct nucleophilic attack on the phosphoryl center by hydroxide ion and the increase in hydrolytic stability with progressive nitrogen substitution can be accounted for by less favorable polar and steric effects.
        
1 lessTitle: Juvenile hormone esterase (JHE) from Tenebrio molitor: full-length cDNA sequence, in vitro expression, and characterization of the recombinant protein Hinton AC, Hammock BD Ref: Insect Biochemistry & Molecular Biology, 33:477, 2003 : PubMed
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 (66000) and pI (4.9) as was expected from the deduced amino acid sequence of the cDNA.
        
Title: In vitro expression and biochemical characterization of juvenile hormone esterase from Manduca sexta Hinton AC, Hammock BD Ref: Insect Biochemistry & Molecular Biology, 33:317, 2003 : PubMed
Juvenile hormone esterase (JHE) is a selective enzyme that hydrolyzes the methyl ester of juvenile hormone. This enzyme plays an important role in the regulation of metamorphosis in caterpillars, and is implicated in additional roles in development and reproduction in this and other orders of insect. The full length coding region of the JHE cDNA from Manduca sexta was subcloned into the baculovirus AcMNPV genome under the control of the p10 promoter. The recombinant virus demonstrated the expression of high levels of JHE activity when infected into Hi5 cells from Trichoplusia ni. The recombinant protein was partially purified by anion exchange chromatography and its biochemical characterization showed similar features to the wild type protein. The recombinant JHE has an estimated MW of 66500 Da. Some heterogeneity with the enzyme was observed when analyzed by isoelectric focusing, although the peak of JHE activity was observed at pI=6.0. It is highly sensitive to trifluoroketone inhibitors and certain phosphoramidothiolates, while relatively insensitive to other common esterase inhibitors. Incubating the enzyme with various organic solvents and detergents showed that the enzyme is activated at lower concentrations of solvents/detergents and remains significantly active even at high concentrations. The high tolerance of organic solvents may make this JHE enzyme useful in future applications as a synthetic catalyst.
        
Title: The role of low levels of juvenile hormone esterase in the metamorphosis of Manduca sexta Browder MH, D'Amico LJ, Nijhout HF Ref: J Insect Sci, 1:11, 2001 : PubMed
The activity of juvenile hormone esterase (JHE) in feeding fifth instar larvae of Manduca sexta increases gradually with larval weight and rises to a peak after larvae pass the critical weight when juvenile hormone secretion ceases. Starvation of larvae of Manduca sexta (L.) that had exceeded the critical weight inhibited peak levels of JHE, but did not delay entry into the wandering stage when larvae leave the plant in search of a pupation site. This suggests that peak levels of JHE may not be essential for the normal timing of metamorphosis. Starved larvae pupated normally, indicating the peak of JHE was not necessary for a morphologically normal pupation. Treatments of larvae with the selective JHE inhibitor O-ethyl-S-phenyl phosphoramidothiolate (EPPAT) that began immediately after larvae achieved the critical weight (6.0 to 6.5 grams for our strain of Manduca) delayed entry into the wandering stage. By contrast, EPPAT treatment of larvae at weights above 8.0 g had no effect on the subsequent timing of the onset of wandering. Therefore, although the normal timing of the onset of wandering does not require peak levels of JHE, it requires low to moderate levels of JHE to be present until larvae reach a weight of about 8.0 g.
        
Title: Insecticidal, anticholinesterase, and hydrolytic properties of S-aryl phosphoramidothioates Sanborn JR, Fukuto TR Ref: Journal of Agricultural and Food Chemistry, 20:926, 1972 : PubMed
The insecticidal, anticholinesterase, and hydrolytic properties for a series of S-phenyl phosphoramidothioates and 5-phenyl phosphonamidothioates were examined. The compounds were moderately toxic to the housefly and were effective inhibitors of cholinesterase. Attempts to correlate cholinesterase inhibition of housefly toxicity with physical organic parameters were unsuccessful. However, an excellent linear relationship was obtained between Hammett's sigma constant and alkaline hydrolysis rates of the O-ethyl substituted S-phenyl phosphoramidothioates. In addition, a kinetic study of the alkaline hydrolysis of these esters was carried out for the purpose of examining the mechanism of reaction. The results indicate that hydrolysis takes place by a direct nucleophilic attack on the phosphoryl center by hydroxide ion and the increase in hydrolytic stability with progressive nitrogen substitution can be accounted for by less favorable polar and steric effects.