Ecdysteroid receptors in a tumorous blood cell line of <i>Drosophila melanogaster</i>

Abstract The tumorous Drosophila melanogaster blood cell line BII has been studied for evidence for the presence of ecdysteroid receptors. The [ 3 H]ponasterone A (pon A)* used in this study has been extensively purified, and the location of the tritium in the molecule has been partially determined. BII cells do not metabolise ecdysteroids. Intact cells demonstrate a considerable specific uptake of [ 3 H]pon A which is saturable, apparently showing two specific components: a very high affinity component (K D = 0.3 nM) and a high affinity component (K D = 2 nM). The specific binding of [ 3 H]pon A to whole cells is compatible with unlabelled ecdysteroids, but not with mammalian steroid hormones. The association rate constant (k a ) for [ 3 H]pon A was determined to be 3 × 10 7 M −1 min −1 at 21 °C, while the dissociation rate constant (k d ) for the specifically bound [ 3 H]pon A was found to be 4.4 × 10 −3 /min. Together, the kinetic rate constants yield a value of 0.15 nM for the K D . The receptors have been partially characterised in a cell‐free extract prepared by sonification of the cells. The optimum pH for extraction and hormone binding is 8.2. Scatchard plots of binding data indicate that the cell‐free extract also contains two high affinity specific binding components (k D = 0.1 nM and K D = 1 nM). The hgih affinity binders are macromolecular, as shown by chromatography on Sephadex G‐25, and are susceptible to protease digestion, heat, and treatment with N‐ethylmaleimide. Sucrose density centrifugation of the labelled receptor shows one peak at approximately 6S. The stability of the receptor preparation has been studied and conditions have been empirically determined (10% w/v sucrose, 25 mM dithioerthreitol, and 10 mM citrate), whereby the binding capacity of the unlabelled receptor is stable for at least 8 weeks if frozen at −20°C.

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