In vitro ecdysteroid conjugation by enzymes of Manduca sexta midgut cytosol

Abstract

In incubations with 80,OOOg supernatant of Manduca sexta midgut homogenates, [3H]ecdysone was converted to 3-[3H]epiecdysone and tritiumlabeled highly polar metabolites. CI8 SEP-PAK cartridges were found suitable for the separation and purification of the free ecdysteroids and of the highly polar metabolites. Eighty to ninety percent of the metabolites were hydrolyzed by enzyme mixtures (mainly 0-glucuronidase, sulphatase, and acid phosphatase) from molluscs, even when 0-glucuronidase activity was completely inhibited by D-saccharic acid 1,4lactone, or various human acid phosphatases (free of sulphatase activity). In each experiment, the hydrolysate contained a much higher proportion of 3-epiecydsone than the free (unconjugated) ecdysteroid fraction. [3H]ecdysone was not metabolized in anaerobic incubations of midgut supernatant that had been filtered through Sephadex C-25. Addition of 5 m M ATP and 5 m M Mg2+ restored the conjugate formation in incubations of Sephadex-filtered supernatant. Four ecdysone conjugates and two 3-epiecdysone conjugates were resolved by reversedphase ion-pair high-performance liquid chromatography. It i s concluded that the midgut cytosol contains several ATP:ecdysteriod phosphotransferases. This is the first demonstration of the formation of ecdysteroid phosphoconjugates in a cell-free system.

Induction of Enzymes Involved in Molting Hormone (Ecdysteroid) Inactivation by Ecdysteroids and an Agonist, 1,2-Dibenzoyl-1-tert-butylhydrazine (RH-5849)

“…This indicates that the corresponding 3-epiecdysone phosphotransferase product is 3-epiecdysone 3-phosphate. The identification of 3-epiecdysone 2-phosphate and 3-epiecdysone 3-phosphate as products of the 3-epiecdysone phosphotransferase in M. sexta midgut is consistent with the reported detection of two 3-epiecdysone phosphoconjugates in this system (22).…”

Diversity of detoxification pathways of ingested ecdysteroids among phytophagous insects

“…Manduca sexta Weirich et al (1986Weirich et al ( , 1991 Weirich et al (1986) b Spodoptera littoralis Webb et al (1995Webb et al ( , 1996 b Side-chain cleavage to poststerone Bombyx mori Hikino et al (1971) *Two reactions can be combined, e.g., the double-conjugates in Locusta (3-acetate + 2-phosphate), the 3-epimer + 3-phosphate (Pieris), and the 3-epimer or 3-dehydro + 22-acyl esters (Plodia). a 14-Dehydroxylation takes place after feeding ecdysteroids, but this reaction is most probably due to gut bacteria and not to insect tissues.…”

“…; Lafont (1994) Manduca sexta Weirich et al (1986) b Spodoptera littoralis Webb et al (1995Webb et al ( , 1996 b 3-dehydroDrosophila melanogaster Sommé-Martin et al (1988) …”

Analysis of Free and Conjugated Ecdysteroids and Polar Metabolites of Insects

“…4. When ecdysone and 20-hydroxyecdysone are applied to a C 18 Sep-Pak and then eluted with 5 ml each of 30, 60, and 100% methanol, the ecdysteroids are eluted with 60% methanol (Thompson et αί, 1985c;Weirich, 1985;Weirich et al, 1986). 2) and fractions 2 and 3 ( Fig.…”

“…PI-RP-HPLC on a radial compression C 18 column has also been used to resolve five different conjugates from M sexta midgut supernatant incubated with [ 3 H]ecdysone (Weirich et al, 1986). …”