In vitro ecdysteroid conjugation by enzymes ofManduca sexta midgut cytosol

In incubations with 80,000g supernatant of Manduca sexta midgut homogenates, [3H]ecdysone was converted to 3-[3H]epiecdysone and tritiumlabeled highly polar metabolites. C18 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 β-glucuronidase, sulphatase, and acid phosphatase) from molluscs, even when β-glucuronidase activity was completely inhibited by D-saccharic acid 1,4-lactone, 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 G-25. Addition of 5 mM ATP and 5 mM 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 is 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.