Juvenile hormone esterase activity from Manduca sexta corpora allata in vitro

Abstract

Juvenile hormone esterase (JHE) activity released by the corpora allata (CA) into incubation media (CA-JHE) was titered daily during the course of the last (fifth [V]) larval stadium of Manduca sexta. This CA-JHE activity was relatively low during the early last stadium up to the time of commitment (V4), then rose rapidly to a peak on V6. Activity declined sharply almost to precommitment levels by V8, before rising to a second peak o n the first day of the pupal phase (PO). This pattern of activity i s distinct from that of hemolymph JHE activity, which peaks just prior to wandering o n V4 and again just prior to pupation (V9). Although the CA-JHE and hemolymph-JHE possess different temporal patterns of activity, isoelectric focusing, gel electrophoresis, and initial studies with selected inhibitors suggest that the enzymes responsible for the CA-JHE and hemolymph-JHE activities are similar, but not identical, in nature.Exposure of the V6 CA in vitro to JH II (0.1 pM) or fluoromevalonolactone (FMev; 0.1 mM) produced an approximate fivefold increase and 60% decrease in JH acid synthesis, respectively. Conversely, the same treatments resulted in an inhibition (JH II) and stimulation (FMev) of CA-JHE activity. These observations suggest that JH may be involved in the direct positive feedback regulation of postwandering larval CA and that the CA-JHE may also be integrally related to this positive feedback mechanism.Key words: endocrine feedback, hemolymph JH esterase, fluoromevalonolactone Acknowledgments: The authors thank Louise Studley for rearing the insects, Charles Allen and William Janzen for their help with the dissections, and Jay P. Woodring, Abner Hammond, Tad Hardy, and R. Mike Roe for their very useful discussions and manuscript comments.

Effects of endogenous esterases and an allatostatin on the products of Manduca sexta larval corpora allata in vitro

“…Hydrolysis of newly synthesized JH to J H acid would also affect the interpretation of the product synthesized at these times, if the assay used did not discriminate between the two compounds. The elevated level of JH esterase activity from the CA on day 0 of the fifth stadium is not reflected by either the haemolymph J H esterase activity (Sparks et al, 1983), which is very low at the beginning of the stadium, nor by esterase activity from the CA determined under the appropriate conditions of substrate saturation (Sparks et al, 1989). However, significant levels of esterase activity were observed previously in incubations of CA from day 0 fifth instars, when the JH esterase activity of the glands was measured by the direct addition of a small amount of radiolabelled JH, far below substrate saturation, to incubation medium containing CA (Granger et al, 1986).…”

“…As a means of assessing the contribution of the endogenous CA esterases to JH acid in the incubation medium, the effect of the potent JH esterase (JHE) inhibitor Sbenzyl-O-ethyl phosphoramidothiolate (BEPAT) (Sparks et al, 1983) on the levels of JH and JH acid in CA incubations was determined with the partition-RIA procedure by comparison of these levels in incubations of CA with and without BEPAT. Utilizing the assay for JH esterase activity described by Hammock & Sparks (1977), a concentration of M BEPAT was found to inhibit greater than 85% of the JH esterase activity in medium from incubations of CA from days 6 and 7 of the fifth stadium, glands which have been shown previously to have the highest levels of endogenous esterase activity in this stadium (Sparks et al, 1989). At lop6 and M BEPAT, inhibition was greater than 93%.…”

“…JH esterase, which in the haemolymph hydrolyses circulating active hormone to the acid at critical times during the last (fifth) larval stadium of Manduca (Vince & Gilbert, 1977;Sparks et al, 1983;Hammock, 1985;Roe & Venkatesh, 1990), has been measured in a variety of Manduca tissues (R. M. Roe, personal communication). Substantial activity has been found in incubations of CA from different stages of fifth stadium Manduca larvae (Sparagana et al, 1984;Granger et al, 1986), the most recent study showing a single major peak of activity with CA from day 6 of this stadium (Sparks et al, 1989). Since the product of the CA after day 4 is JH acid, any esterases present in incubations of CA after this time would not affect the measurement of JH synthesis.…”

“…However, the contribution of esterase activity to the measured levels of JH and JH acid in incubations of CA between days 0 and 4 is not known. The inclusion of an esterase inhibitor in incubations of CA, followed by the separation of the JH and JH acid prior to assay, permits a direct determination of the contribution of endogenous esterase activity to the JH and JH acid levels measured on each day and provides a basis for comparison with published quantitative measurements of esterase activity released by CA in vitro (Granger et al, 1986;Sparks et al, 1989). Furthermore, these same data more precisely define the time over which the biochemical switch from JH to JH acid production occurs.…”

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

“…These two forms of JHDPT may both be present in vivo, or, alternatively, one of them may have arisen artifactually during sample processing. Multiple electrophoretic variants have been reported for the JHE from a single tissue type of this species (Jesudason et al, 1990(Jesudason et al, , 1992Sparks et al, 1989) and several other lepidopterans (e.g., Abdel-Aal et al, 1988;Hammock, 1985;Hanzlik and Hammock, 1987). The use of electrophoretically purified JHDPT indicates a strict dependence upon ATP and Mg2+ for activity.…”