Novel assay for determining the metabolic fate of juvenile hormone III: A study with Drosophila melanogaster

Abstract

A thin-layer chromatographic assay was developed for the resolution of hydrolytic and conjugative catabolites of juvenile hormone (JH). A singledimension, dual-development thin-layer system allowed complete resolution of t h e catabolites. Thus, this system provided a means for the rapid and economic analysis of J H hydrolysis even when different hydrolytic activities were present concurrently. Purified hydrolytic enzymes were found to be superior to chemical methods for the generation of small amounts of standards of J H catabolites. The relative levels of activities of an epoxide hydrolase and an esterase toward JH I l l were found to be similar in microsomal preparations from three lines of adult Drosophila rnelanogaster isolated from a field population. However, selection of flies by exposure to cut orange resulted in the elevation of levels of epoxide hydrolase activities, whereas esterase levels were not affected to the same extent. The formation of the J H acid-diol was not detected under the conditions of this study, suggesting that the J H acid and diol were not good substrates for epoxide hydrolase and juvenile hormone esterase, respectively.The JHs* are terpenoid epoxides that are central regulators of insect biology [l-31. The JHs are directly involved in the control of a variety of events critical for the survival of insects including metamorphosis [4], caste determination [5], diapause [6], and vitellogenesis [7-lo].Perturbations in JH levels at critical times during development have dramatic effects on both form and function in insects [11][12][13][14]. Thus, insecticides

Effect of mating on plasma juvenile hormone esterase activity in females of Choristoneura fumiferana and C. rosaceana

“…The JH metabolites produced by incubation of synthetic JH I11 with plasma from the two tortricids were identified using the TLC method of Hammock and Roe (1985). Radiolabeled metabolite standards were prepared enzymatically using recombinant JHE and murine EH according to the method of Ottea et al (1988). JH Ill acid made up 93% (C. fumiferana) and 97% (C. rosaceana) of the measurable JH I11 metabolites, thus validating the use of the Hammock and Sparks (1977) partition assay for the measurement of JHE activity in our two systems.…”

Gerry Brooks and epoxide hydrolases: four decades to a pharmaceutical

“…Mullin22, 23 developed a nice hypothesis that epoxide hydrolases in insects may be associated with the epoxide functionality being common among plant natural products that are found in their diet. The epoxide hydrolases of Drosophila melanogaster (Meigen) and other flies have been investigated enough to provide a basis for future work 24–27. Possibly, such model species can help to address the endogenous roles of the microsomal enzymes as well as their role in xenobiotic metabolism.…”

Characterization of the juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol phosphotransferase (JHDPT) from Manduca sexta Malpighian tubules

“…In numerous studies, JHE activity has been associated with either hemolymph or soluble tissue fractions, though its presence (contamination?) in membrane fractions has also been either reported or implied in the data presented (Kramer et al, 1977;Wing et al, 1981;Ottea et al, 1988;Casas et al, 1991a;Jesudason et al, 1992;Campbell et al, 1992). These studies also indicate that JHEH is a membrane-bound enzyme, though several reports of JHEH activity associated with cytosolic fractions have appeared (Wisniewski et al, 1986; Casas et al, 1991a,b;Harshman et al, 1991;Jesudason et al, 1992;Campbell et al, 1992).…”

“…The presence of very polar metabolites of labeled JH has often been either reported or implied in data presented (White, 1972;Ajami and Riddiford, 1973;Slade and Wilkinson, 1974;Erley et al, 1975;Edwards and Rowlands, 1978;Kramer et al, 1977;Wilson and Gilbert, 1978;Halarnkar and Schooley, 1990;Halarnkar et al, 1993;Zera et al, 1993), but their absence has also been observed using Musca microsomes (Yu and Terriere, 1978b); Dvosophila microsomes (Ottea et al, 1988); crude homogenate of M . sexta midgut, integument, and fat body Uesudason et al, 1992); and M. sexta embryo homogenates .…”

“…Thus far, we have been unable to obtain l3H1JH I acid-diol-phosphate by incubating either [3H] JH I diol-phosphate with hemolymph or by incubating [3H]JH I acid-diol with Mt (data not shown). The lack of very polar JH metabolite generation by dipteran microsomal fractions is not surprising (Yu and Terriere, 1978b;Ottea et al, 1988) in light of the exclusively cytosolic nature of M . sexta Mt JHDPT indicated in this study.…”