Binding Proteins for an Ecdysone Metabolite in the Crustacean Hepatopancreas

Abstract

When crustacean hepatopancreas is incubated in the presence of a-[3Hlecdysone of high specific activity and is then homogenized and centrifuged, a peak of protein-radioactivity is recovered after gel filtration of the 105,000 X g supernatant. Analysis of this peak by sucrose gradient centrifugation revealed the presence of two complexes of protein and labeled material (-11.5 S and 6.35 S). The same results were obtained in vivo. On standing at low ionic strength, the lighter component disappeared, suggesting that the heavier component is an aggregate of the lighter one. Chemical analysis of radioactive material in the complex revealed that it is not a-or j3-ecdysone nor any previously described metabolite of the ecdysones.This new metabolite of a-ecdysone is found mainly in the incubated hepatopancreas. Partial structures consistent with the analytical data are inferred for this metabolite. It is suggested that the metabolite may be active in the action of molting hormone.Although highly significant advances regarding the chemical nature of arthropod growth hormones have occurred in recent years (1, 2), we do not yet fully understand how these molecules elicit their effects. This lack of understanding is due in part to a paucity of information regarding the primary step in hormone action, i.e., the means by which the hormone recognizes the target tissue (or the converse) and is "bound" to cell constituents. The existence of proteins that bind steroid hormones is an established fact in several mammalian systems (3, 4), and progress in that field is due in great part to the synthesis of labeled hormones of high specific activity. The synthesis of tritiated a-ecdysone of high specific activity (5) provided the critical tool for studies on the binding of molting hormone to arthropod target systems. We now know that a-ecdysone is rapidly converted to ,3-ecdysone (ecdysterone, 20-hydroxyecdysone, crustecdysone) by insects and crustaceans (6, 7), and it is believed that j3-ecdysone is the molting hormone of crustaceans (8-11). The present study examines the possibility of hormone-binding macromolecules in the crayfish hepatopancreas, an organ analogous to the vertebrate liver and the insect fat body. MATERIALS AND METHODSThe a-[23,24-8H]ecdysone was synthesized by catalytic tritiation of 2,,3(3,14a,22,,25-pentahydroxy-5(3-cholest-7-en-23-yn-6-one (5). Male crayfish (Orconectes virilis) were cooled on ice for 30 min, and the hepatopancreas was removed. The hepatopancreas was rinsed in crayfish saline (12) and incubated at 250 in 3 ml of saline containing [8H]ecdysone (see figure legends for quantities). After incubation, the tissue was rinsed twice in cold 0.1 M phosphate buffer (pH 7.3) and homogenized in buffer in a ground glass homogenizer at 4°. After centrifugation of the homogenate at 105,000 X g for 1.5hr at 40, the supernatant was concentrated with lyphogel and aliquots of the concentrated supernatant were assayed for protein (13) The hepatopancreas is rich in lipid (14, 15), which necessitated partial...

Selective Binding of [ 3 H]Gibberellin A 1 by Protein Fractions from Dwarf Pea Epicotyls

“…The behavior of the hormone-receptor complex, subsequent to binding, has been clearly characterized in the uterine response to estradiol (1). Similar data have been obtained on the association between a metabolite of a-ecdvsone and the crustacean hepatopancreas receptor (2).…”

Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus

“…Here the enzymatic activity of cuticular hemocyanin was higher than that of hemocyanin derived from the hemolymph [ 53 ]. Additionally, ecdysone (arthropod moulting hormone) has been found to bind to proteins within the crustacean hepatopancreas and cuticle [ 54 ]. More recent studies on the tarantula, suggest that this protein may be hemocyanin.…”