Ecdysteroid receptors in a tumorous blood cell line of Drosophila melanogaster.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 2:295-317 (1985) Ecdysteroid Receptors in a Tumorous Blood Cell Line of Drosophila melanogaster Laurence Dinan lnstitut fiir Zoologie der TechnischenHochschule Damstadt, Schnittspuhnstrusse 10, 0-6100 Dumstadt and lnstitut fiir Zoologie 111, Universitat Dusseldorf, Universitatsstrasse1, D-4000 Dusseldorf 7, Federal Republic of Germany The tumorous Drosophila melanogaster blood cell line BII has been studied for evidence for the presence of ecdysteroid receptors. The 13H]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. Bll cells do not metabolise ecdysteroids. Intact cells demonstrate a considerable specific uptake of [3H]pon A which is saturable, apparently showing two specific components: a very high affinity component (KD = 0.3 nM) and a high affinity component (KD = 2 nM). The specific binding of [3H]pon A t o whole cells is compatible with unlabelled ecdysteroids, but not with mammalian steroid hormones. The association rate constant (kJ for [3H]pon A was determined t o b e 3 x 107M-’min-’ at 2I0C, while the dissociation rate constant (kd) for the specifically bound [3H]pon A was found to be 4.4 x 10-3/min. Together, the kinetic rate constants yield a value of 0.15 n M for the KD. The receptors have been partially characterised in a cell-free extract prepared by sonification of the cells. The optimum p H for extraction and hormone binding i s 8.2. Scatchard plots of binding data indicate that the cell- *Abbreviations: BSA = bovine serum albumin; DCC = dextran-coated charcoal; DTE = dithioerythreitol; EDTA = sodium ethylenediamine tetraacetate; HEPES = 4(2-hydroxyethyl)I-piperazineethane sulphonic acid; HPLC = high-performance liquid chromatography; hptlc = high-performance thin-layer chromatography; H-R complex = hormone-receptor complex; k, = association rate constant; kd = dissociation rate constant; KA = equilibrium association constant; KD = equilibrium dissociation constant; MS = mass spectrometry; NEM = Nethylmaleimide; pon A = ponasterone A; Rf = distance migrated relative to solvent front; RIA = radioimmunoassay; t% = half-life; tlc = thin-layer chromatography; tris = tris(hydroxymethy1)methylamine. Acknowledgments: The work reported in this paper was supported by the Deutsche Forschungsgemeinschaft with grants to Professor Emmerich and Professor Spindler. During the major part of this work (in Darmstadt), the author was the recipient of a Royal Society Overseas Fellowship, which he gratefully acknowledges. He should also like to thank Professor Ernmerich and Professor Spindler for providing facilities, Professor Spindler, Drs. G . Gellissen and M. Londershausen for their comments on the manuscript, Dr. H.H. Rees for the MS of poststerone, and Professor E. Gateff for providing the BII cell line. The RIA was kindly performed by M. Schwab. The synthesis of [3H]ponasteroneA was funded by the Deutsche Forschungsgemeinschaft. Laurence Dinan is now at the Department of Biological Sciences, University of Exeter, Exeter, Devon, United Kingdom; address reprint requests there. Received July 19,1984; accepted December 20,1984. 0 1985 Alan R. Liss, Inc. 296 Dinan free extract also contains two high affinity specific binding components (KD = 0.1 n M and KD = 1 nM). The high affinity binders are macromolecular, as shown by chromatography on Sephadex (2-25, 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 dithioerythreitol, and I0 m M citrate), whereby the binding capacity of the unlabelled receptor is stable for at least 8 weeks if frozen at - 2 O O C . Key words: ecdysteroids, steroid hormone receptors, Drosophila cell line, haemocytes INTRODUCTION Persuasive evidence for the existence of steroid hormone receptors in insects has only been recently obtained [1,2].All subsequent work on steroid receptors in insect systems has concentrated on one species, Drosophila melanogaster, using imaginal discs ,salivary glands , and the K, cell line [1,461. Cell lines offer several advantages for the study of steroid receptors. It is relatively easy to obtain large quantities of homogeneous material, and also, the cell line contains no endogenous hormone (if the cells themselves do not produce the hormone). A large number of DrosophiZa cell lines exist; several of these are known to respond to ecdysteroids [7l and are, therefore, good candidates for studies on ecdysteroid receptors. However, almost all these cell lines have been derived from dissociated embryos, and it is not known which, if any, in vivo cell type is represented by the cell line. Two Drosophila cell lines of defined origin do, however, exist. They were derived from tumorous blood cell mutants . Mutant larvae develop normally until the third instar when the normal increase in the number of haemocytes that occurs in wild-type larvae becomes uncontrolled in the mutants, resulting in the production of between 20- and 200-fold more haemocytes. Additionally, these mutant blood cells cannot recognise self from non-self; they digest not only the histolysing larval tissues, but also all other tissues that should be carried over into the pupal and adult stages . In cell culture the blood cells respond to the presence of ecdysteroids by increased phagocytosis and lysis, which results in a declining cell density and an enlargement of the remaining cells . In this report, the initial results concerning the demonstration of the specific uptake and binding of ecdysteroids by one of these two tumorous blood cell lines are presented. Since this is the first report using this batch of radiolabelled ponasterone A* for receptor studies, the purification and characterisation are reported in some detail. *Trivial names: Ecdysone = 20, 3f3,14or, 22R, 25-pentahydroxy-5-cholest-7-en-6-one; 20-hydroxyecdysone = 2&3& 14a,20R, 22R, 25-hexahydroxy-5cholest-7-end-one; ponasterone A = 20, 3f3,14a, 20R, 22R-pentahydroxy-5-cholest-7-en-6-one. Ecdysteroid Receptors 297 MATERIALS AND METHODS Preparation and Purification of [3H]Ponasterone A The synthesis of [3H]pon A was carried out as a collaborative project by the research groups of Professor Emmerich (Technische Hochschule, Darmstadt), Professor Koolman (Philipps-Universitat, Marburg), and Professor Spindler (Universitat Dusseldorf). 20-Hydroxyecdysone (Fig. 1, 11) was dehydrated by the method of Hein(IV). However, rich  to yield 25-deoxy-26-dehydro-20-hydroxyecdysone reverse-phase HPLC separation of the product showed that it consisted of two components. NMR and MS indicate that the product probably contained 24-dehydro-25-deoxy-20-hydroxyecdysone (V) in addition to the expected product. This mixture (35 mg) was tritiated with tritium gas by NEN (Dreieich, FRG). The crude ponasterone A was purified by column chromatography on silicic acid to a specific radioactivity of 13.2 Cilmmol. However, the reactants and products of the tritiation possess almost identical Rf values on tlc, and they would not have been separated under the conditions used. This separation was achieved by reverse-phase HPLC (Column: Waters Partisil ODs, 25 cm X 4.6 mm i.d. Solvent: methanollwater, 6:4.Flow rate: 0.5 mllmin. Retention times: pon A, 19.5 min; starting compounds, 15.5 and 16.5 min). The column had previously been calibrated by the injection of known quantities of a standard solution of pon A, and it was therefore possible to calculate the specific radioactivity of the purified [3H]pon A (three determinations: 182, 182, and 186 Cilmmol). Radioassay indicated that the recovered reactants were not radioactive. Hptlc of the purified [3H]pon A (Merck Kieselgel 60 F254 plates; solvent, CH2C12/EtOH,9:1, run three times) showed that it contained a 12% radioactive impurity of the same specific radioactivity, which was slightly less polar than pon A. This impurity was separated by normal-phase HPLC (Column: Merck Lichrosorb Si 60, 5 pm, 25 cm x 4.6 mm i.d. Solvent: CH2C12l isopropanollH20, 250:50:3. Flow rate: 0.5 mllmin. Retention times: pon A, 21 R HHO o d 0? R=. I. IV . Fig. 1. Ecdysteroid structures. I, Ponasterone A; II, 20-hydroxyecdysone; Ill, ecdysone; IV, 25deoxy-26-dehydro-20-hydroxyecdysone; V, 24-dehydro-25-deoxy-20-hydroxyecdysone; VI, poststerone. Dinan 298 min; impurity, 17.5 min.). The highly purified [3H]pon A was stored in methanollbenzene (9:l) at 4°C. Stability of [3H]Pon A The radiochemical purity of the [3H]pon A was tested regularly by hptlc and reverse-phase HPLC. As an example, the results obtained 2 years after the synthesis of the [3H]pon A are shown in Figure 2. Hptlc of the crude [3H]pon A preparation reveals little degradation after this time. In contrast, reverse-phase HPLC shows that only approximately 50% of the radioactivity is still in the form of pon A. Fortunately, all the radioactive degradation products elute from the reverse-phase column before pon A, and this system can be used to remove the degradation products. Determination of the Location of 3H in the Purified [3H]Pon A The very high specific radioactivity of the purified [3H]pon A indicates that an average of six tritium atoms has been introduced per molecule of pon A during the reductive titiation. It was therefore of interest to determine where those tritium atoms are located. Since the unreacted starting compounds recovered after the tritiation were not radioactive, this would suggest that the tritium atoms are all located in the side-chain (ie, no nonspecific tritiation has taken place). This was shown to be the case by the chemical side-chain cleavage between C-20 and C-22 of the [3H]ponA with pyridinium chlorochromate according to the method of Nali and Rees (personal communication). Carrier pon A (2 mg) was mixed with [3H]p~nA (3 x lo5 dpm), dissolved in 150 pl anhydrous pyridine, and cooled to - 20°C for 30 min. Pyridinium chlorochromate (3 mg) was added to the cold solution and the mixture was stirred at room temperature for 3.5 h. The mixture was partiB - 1 t f PON A 7 6 4- z . 0 I2m 1 0 2 4 6 ' MIGRATION DISTANCE 8 ' [CM] OJ 0 15 20 ELUTION TIME [MINI 5 10 Fig. 2. Radiochemical purity of [3H]pon A 2 years after its synthesis. A small aliquot was removed from the stock solution and unlabelled pon A was added as carrier. The radiochemical purity was then tested by A) hptlc (solvent; CH,CI,/MeOH, 41 v/v) and B) reverse-phase HPLC (solvent; MeOH/H,O, 1:l v/v. Flow rate; 1 ml/min, isocratic elution). The shoulder on the pon A peak in the hptlc scan i s the radioactive impurity, which can be separated by normal-phase HPLC. The arrows show the positions of standard ponasterone A (pon A) and 20-hydroxyecdysone (0). SF, solvent front; 0, origin. Ecdysteroid Receptors 299 tioned between 5 ml each of countersaturated butanol and water. The aqueous phase was further extracted with 5 ml butanol, and the combined butanol phases were washed twice with 5 ml water. After rotary evaporation, the residue from the butanol phases was adsorbed onto Celite and applied to a silica column according to the method of Dinan and Rees [lo]. The 30% methanol in chloroform fraction from the column (containing free ecdysteroids) contained only 6% of the original radioactivity, and analysis by reversephase and normal-phase HPLC showed that the main UV-absorbing compound in this fraction co-chromatographed with poststerone (VI). The identity of this peak was further demonstrated by electron-impact mass spectrometry: mlz 362 YO, M+), mlz 344 (6%, Mf -H20), mlz 250 (57%), mlz 55 (100%). The overall yield of poststerone was 54%, and radioassay of the poststerone eluted from HPLC showed that at least 97% of the tritium had been lost during the side-chain cleavage. Purification of [3H]Ecdysone and Preparation of [3H]20-Hydroxyecdysone [23,24-3H]ecdysone (68 Cilmmol; a gift of the Deutsche Forschungsgemeinschaft) was purified on a 4 g silica column (Merck Kieselgel 60, 70-230 mesh) and eluted sequentially with 40 ml each of 5%, 6%, 8%, lo%, and 15% methanol in chloroform. Five millilitre fractions were collected and analysed by tlc and radioscanning. Fractions containing pure [3H]ecdysone were pooled. A portion of the purified [3H]ecdysonewas converted to [3H]20-hydroxyecdysone by incubation with Malpighian tubules from Locustu rnigruforb according to Feyereisen et a1 [ll].The products were extracted with methanol, partitioned between butanol and water, applied to a 4 g silica column, and eluted with 40 ml each of 5%, looh, 12%, 15%, and 20% methanol in chloroform. Fractions (5 ml) were collected and tested by tlc for ecdysone and 20-hydroxyecdysone. Fractions containing both were re-chromatographed on a second silica column, and fractions containing radiochemically pure [3H]20-hydroxyecdysone were combined with those from the first column. The specific radioactivity of the [3H]20-hydroxyecdysoneproduced was approximately 40 Cilmmol. Purification of Unlabelled Ecdysteroids Pon A was purified by reverse-phase and normal-phase HPLC as described for [3H]pon A. Ecdysone and 20-hydroxyecdysone were purchased from Simes, Milan, and purified by reverse-phase HPLC. Stock solutions were made by dissolving the purified ecdysteroids in methanol and determining the absorbance at 242 nm ( E = 12,000 1lmoUcm). Growth and Isolation of Cells The BII cell line (derived from 1 mbn mutant larvae of D. rnelanoguster) was obtained from E. Gateff (Freiburg, FRG). They were grown in Schneider’s medium supplemented with 10% foetal calf serum as described previously . Cells were isolated by gently shaking the T-flasks or roller bottles 300 Dinan in which they were growing. The cell suspension was then centrifuged (5 min at 350g) in order to pellet the cells. Radioimmunoassay BII cells (2 x lo7 cells) and the medium (10 ml) in which they had grown were extracted with 70% aqueous methanol, and the extracts were tested for using RIA-positive material according to the method of Spindler et a1 [B], the antiserum ICT-1, which has a detection limit of 10-20 pg. No RIA-positive response was detectable in the cells or in the medium. Uptake of [3H]Pon A Into BII Cells Cells (3 x lo7) were incubated in 0.5 ml medium with 0.4nM [3H]pon A (with or without 50 nM unlabelled pon A) at 7°C or 22°C for various times. Cells were then cooled on ice and diluted with 8 ml cold medium. After 5 min, the cells were pelleted (2,0008 for 5 min at 0°C). Aliquots of the supernatant were radioassayed. The cell pellet, after resuspension in 1 ml distilled water, was also radioassayed. Association kinetics were determined with a mini-assay in order to conserve radioligand. The labelled hormone (2.7 x lo6 dpm in 10 pl ethanol/ benzene, 9:1, with or without a 200-fold excess of unlabelled pon A in methanol) was added to 2-ml Eppendorf vials and carefully evaporated under a stream of nitrogen. The hormone was dissolved in 100 pl Schneider's medium and then brought to the required temperature for the experiment (21°C or lT).The cell suspension (1ml, 1.7 x 10' cellslml medium) was also brought to the required temperature and added to the hormone solution, and the vials were quickly vortexed. Aliquots (50 11.) were removed at regular intervals, placed in ice-cooled Eppendorf vials (2 ml), and diluted immediately with 2 ml ice-cold Schneider's medium. After 5 min, the cells were pelleted (1min in a Hettich Mikroliter centrifuge at 4°C). The supernatant was removed by suction and the pellet was washed with a further 2 ml cold medium. The cell pellet was then resuspended in 100 pl distilled water and radioassayed after the addition of 2 ml Aqualuma. The maximal specific binding was determined by incubating an aliquot of the cell suspension with the hormone under the above conditions for 2 h at 21°C and determining the amount of hormone taken up as described above. The saturability of the uptake of pon A was determined by incubating 3 x lo7 cells in 0.5 ml medium with increasing concentrations of [3H]p~n A for 3 h at 22°C in the presence or absence of a 100-fold excess of unlabelled pon A. The uptake was determined as described above, and the results were plotted according to Scatchard . Dissociation of [3H]Pon A From BII Cells The method of Beckers et a1  was used. Cells (1.7 x 108/0.8ml medium) were incubated with 3 nM [3H]pon A (with or without a 200-fold excess of unlabelled pon A) for 3 h at 22°C. The cells were diluted with cold medium and pelleted at 0°C. The medium was removed and replaced with medium (22"C, 0.5 ml; total volume = 0.8 ml) containing unlabelled pon A at the Ecdysteroid Receptors 301 same concentration as the [3H]pon A which had not been taken up, or at a 200-fold excess over the initial [3H]pon A concentration. At known time points thereafter, aliquots (25 pl) were removed, cooled on ice, and diluted with cold medium (1 ml). The cells were pelleted, and the radioactivity associated with the cell pellet was determined by radioassay. Sonification of the Cells and Preparation of the Cell-Free Extract The cell pellet was resuspended in buffer (75 mM TrislHCl, pH 8, or 10 mh4 HEPES buffer, pH 8, both containing 160 pglml aprotinin) to a density of 10' cellslml and sonified at 4°C with one 15s burst (Branson Sonic Power Co., Danbury, CT, USA; setting 1, 40 W). The homogenate was centrifuged for 30 min at 4°C and 20,OOOg, and the supernatant was used for receptor studies. Charcoal Assays Specifically bound [3H]pon A was determined by adsorption of free pon A to dextran-coated charcoal. In the standard assay, 100 pl of the sonification preparation was incubated with 100 pl t3H]pon A (with or without a 200-fold excess of unlabelled pon A) in buffer for 2 h at 25°C. At the end of the incubation, the assay tubes were cooled in ice water, and charcoal suspension (50 pl) was added. The tubes were quickly vortexed and then left to stand at 1°C for 5 min before the charcoal was pelleted (Hettich Mikrohter centrifuge; 2 min at 4°C). An aliquot of the supernatant (125 p1) was removed for radioassay. Two charcoal suspensions have been used during the course of this work. During the initial part of the work, the cells were sonified in Tris buffer and a charcoal suspension (A: 0.5 g acid-washed Norit A charcoal 0.05 g T60 Dextranl20 ml Tris buffer) was added. In the later experiments, the cells were sonified in HEPES buffer, and a more concentrated charcoal suspension (B: 2.5 g acid-washed Norit A charcoal + 0.25 g T60 Dextranl20 ml HEPES buffer) was used, as it was found that this removed a large portion of the nonspecifically bound radioactivity but did not affect the level of specifically bound radioactivity. Specific binding is expressed as the difference between the dpm bound in the absence and presence of excess unlabelled pon A. + Gel-Filtration Chromatography Gel-filtration chromatography was performed at 2°C on a column of Sephadex G-25 Superfine (Pharmacia, Uppsala, Sweden; 7 x 2 cm i.d.), equilibrated with 75 mh4 TrislHCl (pH 8 + 25 mM NaC1). The flow rate was 0.75 mllmin, and the total separation time was 15 min. Sucrose Gradient Centrifugation Sucrose gradient determination of the sedimentation coefficient of the receptor was performed on 10% to 40% sucrose gradients (16 ml in 75 mh4 TrislHCl buffer, pH8) in a Sorvall TV-865B vertical rotor (60,000 rpm, 4 h at 1°C). The sonification preparation was incubated with t3H]pon A (12 nh4 f 302 Dinan 200-fold excess unlabelled pon A) for 2 h at 25°C. Unbound hormone was then removed by charcoal treatment. Samples (0.5 ml; approximately 2 mg protein) were applied to the pre-cooled gradients and overlayered with 0.5 ml silicon oil. After the centrifugation, the gradient was separated into 10 drop ( - 450 pl) fractions and these were radioassayed. The S-value was determined by comparison with known standards: BSA (4.35)and cytochrome C (1.9s).The sucrose concentration of the fractions was determined with a refractometer (Schmidt and Haensch, Berlin, FRG). Protein Determination Protein was determined by the method of Bradford  using BSA (Sigma Chemie, Munich, FRG) as standard. Radioassay Samples were radioassayed in 6-mI mini-vials after the addition of Aqualuma (Baker Chemikalien, Gross-Gerau, FRG). Cell pellets were first resuspended in 1 ml distilled water before addition of the scintillant. When the mini-assay was used, cell pellets in Eppendorf vials were resuspended in 100 pl distilled water before the addition of 2 ml Aqualuma to the Eppendorf vial. Radioactivity was measured in a Packard Tri-Carb 460 CD scintillation spectrometer. Where data are presented in dpm, corrections were automatically made for background, counting efficiency, and chemical quenching. RESULTS Metabolism of Ecdysteroids by BII Cells After a 4-h incubation of BII cells with [3H]pon A at 22"C, the radiolabelled compounds present in the cells and in the medium were analysed by hptlc, normal-phase HPLC, and reverse-phase HPLC. No metabolism could be detected by any of the separation techniques used. Since the uptake of [3H]ecdysoneor [3H]20-hydroxyecdysoneinto BII cells is low, cells and medium were extracted together with 6 h incubation at 22°C with the labelled hormone. Hptlc analyses showed that there was no conversion of the labelled ecdysone to 20-hydroxyecdysone or any other metabolite. Similarly, no metabolism of [3H]20-hydroxyecdysonecould be detected. Saturability of the Uptake of [3H]Pon A by BII Cells Preliminary experiments to determine the time-course of the specific uptake of [3H]pon A (0.4 nM) by BII cells showed that equilibrium was reached between 90 and 120 min at 22"C, and after 9 h at 7°C. Comparison of the uptake of [3Hlpon A with that of [14C]inulin (which cannot enter cells) showed a large amount of tritium associated with the cell pellet after 75-min incubation at 22"C, but a very low level of 14C. On washing the cell pellet at 0°C to 4°C with medium, most of the 14Cradioactivity was removed from the cells, whereas the amount of specifically bound 3H remained constant, thus showing that the r3H]pon A is not just loosely adsorbed to the cells. Ecdysteroid Receptors 303 The dependence of the specific uptake on pon A concentration is shown in Figure 3. When these data are plotted according to Scatchard , a plot with two components is obtained (Fig. 3A). The point of inflexion corresponds to the point at which the high specific activity [3H]ponA was diluted with unlabelled pon A to reach the required concentration. The experiment was repeated, but this time an extra series of assay tubes was added in which the required concentration of pon A was obtained with [3H]pon A alone (Fig. 3C, D). Association Kinetics The association kinetics of [3H]ponA (6.75 nM) have been studied at 21°C and 1°C (Fig. 4A,B), and they show second order kinetics (Fig. 4, insets). At 21°C the association rate constant, determined from the slope in Fi (inset), is 2.6 x 1O7M-'min-', while at 1"C, k, is 9.6 X 105M-'min- . 4A gure Dissociation of [3H]Pon A From BII Cells Cells which had been equilibrated in the presence of 3 nM [3H]pon A were isolated and resuspended in the same volume of medium containing unlabelled pon A either at the same concentration as the unbound [3H]pon A  or at 200 times the initial [3H]pon A concentration (ie, 0.6 pM). The latter experiment was performed as a check on the former experiment because it Q 2 LL \ Bound [pM] Bound [pM] Fig. 3. Dependence of binding of pon A by intact cells on the hormone concentration at 22OC. Cells (3 x 107/0.5ml) were incubated for 3 h in the presence of increasing concentrations of 13H]pon A in the presence or absence of a 100-fold excess of unlabelled pon A to correct for nonspecific binding. [3H]pon A concentrations were obtained by using increasing amounts of radiolabelled ligand (180 Cilrnmol, 0 ) or by adding increasing amounts of unlabelled pon A to a constant amount of [3H]pon A (B). Only the data for the specific component of the binding are shown. B and D show plots of the bound pon A vs the included concentration of pon A. A and C give the corresponding Scatchard plots. The dotted lines in C are the resolution of the Scatchard plot, where the total binding was determined with undiluted (3H]ponA into two linear components. 304 Dinan “1 1 - 1 1 I 2 2 3 4 5 MINUTES 6 7 8 3 4 5 6 Incubation Time I 9 7 6 [minl 9 120 I I 5 I s 10 15 20 2 5 30 MINUTES . 10 is 35 do 3’0 i s 40 20 2 5 Incubation Time [mini Fig. 4. Determination of the association rate constant for [3H]pon A (6.75 nM) with intact BII cells at A) 21OC and B) 1OC. The uptake was measured as described in Materials and Methods. nonspecific binding (m); specific binding (0). The data for the Total dpm bound by cells (0); specific binding show second-order kinetics (insets). The gradients were determined by linear regression (r = A, 0.964; €3, 0.982). [R,], initial receptor concentration; [H,], initial free hormone concentration; [R], free receptor concentration at stated time point; [HI, free hormone concentration at stated time point. was considered that the unlabelled pon A concentration may not have been sufficient to compete with the labelled pon A as it dissociated from the cells, owing to the lower affinity of the cells for unlabelled pon A than for the labelled pon A used in this study. The dissociation of [3H]pon A from BII cells obtained at 20°C after the replacement of the unbound [3H]pon A by a 200-fold excess of unlabelled pon A is shown in Figure 5. There are two main components to the dissociation: a rapid initial dissociation (kd = 8 x 10-2/min)owing to the dissociation of the nonspecifically bound t3HIpon A, and a slower component owing to the dissociation of the specifically bound C3H]pon A. The dissociation rate constant for this slower component is 4.4 x 10P3/min.The value obtained when the unbound [3H]pon A was replaced with the same concentration of unlabelled pon A was 3.9 x 10P3/min(data not shown). Competition of the Uptake of [3H]Pon A With Other Ecdysteroids Aliquots of 1.7 x lo7 cells in 100 pl medium were incubated for 4 h at 22°C with 2.5 nh4 [3H]pon A in the presence of various concentrations of other Ecdysteroid Receptors 305 I 20 40 60 Incubation Time 80 100 120 140 [minl Fig. 5. Dissociation of [3H]pon A from BII cells. Cells were incubated with [3H]pon A (3 nM) with or without a 200-fold excess of unlabelled pon A for 3 h at 21OC. The cells were then isolated and resuspended in medium containing 0.5 KM unlabelled hormone. Aliquots (25 PI) were removed at fixed time points, diluted with ice-cold medium, centrifuged, and the cell pellet was radioassayed. Total binding (0);nonspecific binding (m); specific binding (0). The inset shows the data for the specific binding plotted as a pseudo-first order reaction. unlabelled steroids. The amount of radioactivity associated with the cells was determined as described for the association kinetics and is expressed as a percentage of that obtained when the cells were incubated with [3H]pon A alone (Fig. 6 ) . Of the steroids tested, unlabelled pon A (50% competition at 2 nM) was the most effective competitor, followed by 20-hydroxyecdysone (50% competition at 200 nM). The vertebrate steroid hormones, oestradiol, corticosterone, and hydrocortisone, do not compete for the binding, even at 0.1 mM (oestradiol)or 1mM (corticosterone and hydrocortisone). Sonification as a Means of Releasing the High-Affinity Binding Sites From BII Cells After sonification of the cell suspension and centrifugation of the cell debris, the resulting supernatant was tested for its ability to specifically bind [3H]pon A. Preliminary experiments showed that during incubation of the prelabelled preparation with charcoal at 1"C, the amount of specifically bound radioactivity remained constant between 5 and 15 min and that the optimal number of sonifications was one 15-s burst at 40 W. The extent of specific binding, as measured by the charcoal assay, is proportional to the amount of receptor preparation added. The time-course of the uptake of [3H]pon A at 22°C is similar to that obtained with whole cells, equilibrium being obtained after 60-120 min (data not shown). The optimum pH for the extraction and binding of the receptor was determined by resuspending the cells in buffers of different pH values before 306 Dinan Log [Steroid] Fig. 6. Competition of the binding of [3H]pon A by unlabelled steroids. Cells were incubated with radioligand (2.5 nM) for 4 h at 22OC in the presence of increasing concentrations of P-oestradiol (A); unlabelled steroids. Pon A (0);20-hydroxyecdysone (*);ecdysone (0); corticosterone (W); hydrocortisone (A).The cells were diluted with ice-cold medium and pelleted. The radioactivity in the cell pellet was determined and the specifically bound dpm are expressed as a percentage of the specific binding obtained when no unlabelled steroid is present. sonification (Fig. 7A). [3H]Pon A was added to the supernatant after centrifugation in the buffer of appropriate pH, but the charcoal suspension was added in TrislHCl buffer (pH 8). A preliminary experiment showed that charcoal binds free pon A equally well at all pH values between 6.2 and 9.0. The maximum specific binding is obtained at pH 8.2. The effect of various salts on the specific binding is shown in Figure 7B. The dependence of the extent of binding on the hormone concentration is given in Figure 8. These results were obtained by incubating the sonification preparation with increasing amounts of [3H]pon A (180 Ciimmol, with or without a 200-fold excess of unlabelled pon A). Comparison of the results obtained here with those obtained by incubating the same batch of cells (intact)with 9 nM [3H]ponA showed that 60% of the specific binding activity was recovered in the supernatant after sonification. Dissociation of [3H]Pon A From the Ecdysteroid Receptor The receptor preparation was incubated for 2 h at 25°C with [3H]pon A (9 nM 200-fold excess unlabelled pon A) and then cooled to 1°C. Free hormone was removed by adsorption to charcoal (assay B). The rate of dissociation of the bound hormone from the receptor was determined by incubating aliquots of the charcoal supernatant at the required temperature. After known times, the aliquots were incubated at 1°C for 5 min, and then a second charcoal assay was performed to adsorb the dissociated hormone. An aliquot of the supernatant was radioassayed. The dissociation is pseudo-first Ecdysteroid Receptors 307 1 (0 1 E a a v) C .r-7 r x Y E, 0 6.2 6 6 7.0 7 4 7 8 8.2 86 90 Salt Concentration PH [MI Fig. 7. A) Effect of pH on the extractability and binding of the ecdysteroid receptor. BII cells were resuspended in buffers (imidazole/HCI or Tris/HCI; 75 mM) of varying pH, sonified once, and centrifuged. Aliquots of the supernatant were equilibrated with [3H]pon A in the appropriate buffer, and the binding was determined with DCC assay A. Total binding ( 0 ) nonspe; cific binding (m); specific binding (0).6)Effect of salts on the specific binding. Aliquots (100 pl) of the cell extract were incubated for 3 h at 22OC with [3H]pon A (f100-fold excess of unlabelled pon A) in the presence of various salts. NaCl (A);KCI (0); MgCI2 (m); CaCI, (0); MnCI, (0). Results are expressed as a percentage of the specific binding obtained in 75 m M Tris/HCI buffer (pH 8) without the addition of salt. A 2501 9 5 Q n z 3 P [3H-PON A1 nM BOUND PM Fig. 8. A) Saturation of ecdysteroid receptor with [3H]pon A. Aliquots of the cell extract (100 PI)were incubated with 100 pI of buffer containing increasing concentrations of [3H]pon A (180 Ci/mmol; f200-fold excess of unlabelled pon A) for 3 h at 22OC. The amount of bound hormone was determined with DCC assay 6. Total binding ( 0 ) ;nonspecific binding (m); specific binding (0). 6)Data for the specific binding expressed according to Scatchard . 308 Dinan order. The kd is 1 x 10-2/min at 25°C (tv, = 70 min) and 8 x 10-4/min at 1°C (ty2= 15 h). Gel-Filtration Chromatography of the Cell Extract It is possible to demonstrate a specific binding to the cell extract by chromatography on Sephadex G-25 (Fig. 9). Table 1gives the relative amount of label found in the macromolecular fraction after treatment of the extract in various ways, and also, it shows that the amount is considerably reduced by addition of a 100-fold excess of unlabelled pon A, pronase, trypsin, heat, and the addition of NEM or detergents. Sucrose Density Gradient Centrifugation The separation of the prelabelled receptor preparation on 10% to 40% sucrose gradients (Fig. 10) shows one major peak of bound radioactivity at an estimated sedimentation coefficient of 5.8s ( f O . l ; n = 5). This peak is totally absent if the receptor preparation is incubated with [3H]pon A in the presence of a 200-fold excess of unlabelled pon A. Stability of the Receptor Preparation The stability of the receptor in the presence or absence of hormone (9 nM) was studied at 8°C and 25°C (Fig. 11).The receptor (in HEPES buffer + 1 2- 10- 10 20 30 Fraction Number Fig. 9. Demostration of the macromolecular nature of the binding component in the sonification preparation by passage through a gel-filtration column. The preparation was incubated or without ( 0 )a 100-fold excess of unlabelled pon A for 3 h at 22OC. with [3H]pon A with (0) Samples (0.25 ml) were then cooled on ice and separated at 2OC on a Sephadex C-25 Superfine column (7 x 2 cm i.d.) and equilibrated with 75 m M Tris/HCI buffer (pH 8 + 25 mM NaCI; flow rate, 0.75 mllmin). Fractions (10 drops) were collected and radioassayed. Ecdysteroid Receptors 309 TABLE 1. Macromolecular Binding of 13H]Pon A to BII Cell Extract* Percent of control (cpm) in macromolecular peak Incubation conditions r3H]pon A f 100-fold excess unlabelled pon A + pronase 0.6 mglml trypsin 0.4 mglml + RNase 0.4 mglml + DNase 0.4 mglml + heat 5 min at 90°C + NEM 40 mM + Triton X-100 0.8% added with pon A + NP-40 0.8% added with pon A + NP-40 0.8% added at end of incubation 100 2 18 41 70 85 + 2 2 3 2 100 *Radiolabelled pon A (50 pl; 8,000 cpm) was incubated with 150 p1 cell extract for 3 h at 20°C in a total volume of 250 pl. Enzymes included in the incubations were pronase (ex Streptomyces; Serva), trypsin (ex bovine pancreas; type 111, Sigma), DNase (ex bovine pancreas; Serva), and RNase (ex bovine pancreas; Serva). The incubation mixture was cooled on ice and applied to a Sephadex G-25 column as described in Materials and Methods. r40 FRACTION NUMBER Fig. 10. Sucrose gradient density centrifugation of prelabelled BI I ecdysteroid receptor. T h e receptor preparation was equilibrated with 9 nM (3H]pon A with (M) or without ( 0 )a 200fold excess of unlabelled pon A. The preparation was then cooled to IoC and treated with charcoal to remove unbound hormone. Aliquots (0.5 mi) were applied to 10% to 40% sucrose gradients (16 ml), overlayered with 0.5 ml silicon oil, and centrifuged at 60,000 rpm in a vertical rotor for 4 h at 1OC. Gradients were fractionated (10 drops) from the bottom of the gradient. Arrows mark the positions of standard proteins run in a parallel gradient. aprotinin) is considerably more unstable at 8°C in the absence of hormone. In the presence of hormone the specific binding is stable for at least 3 days, whereas in the absence of hormone the specific binding decreases with a half-life of approximately 3 days. At 25°C the decline in specific binding is considerably more rapid, even in the presence of hormone (50% of initial specific binding remains after 20 h). 310 Dinan Y U En v) 1 20 40 60 8b INCUBATION TIME 160 120 [ HOURS] Fig. 11. Stability of the B l l ecdysteroid receptor. The receptor preparation was incubated with 9 n M [3H]ponA (f200-fold excess of unlabelled pon A) for 2 h at 25"C, and then aliquots Alternatively, aliquots were either allowed to continue at 25°C (A) or transferred to 8°C (0). were incubated without hormone at 8OC (W); hormone was then added, followed by an incubation for 2 h at 25°C. Specifically bound hormone was determined with the charcoal assay, and results are expressed as a percentage of the specific binding obtained with freshly prepared cell extract after 2 h incubation with [3H]ponA. The ability of various compounds to improve the stability of the receptor preparation (without hormone) was initially tested by incubating aliquots of the preparation in the presence of the compounds for 3 days at 8°C. Hormone (9 nM k 200-fold excess of unlabelled pon A) was added, and the assays were then incubated at 25°C for 2 h. The specifically bound radioactivity was then determined with charcoal assay B. As a control, the effect of the various compounds on the binding capacity of the fresh preparation was also tested. Of the compounds tested, only molybdate (10 mM) and citrate (10 mM) caused a slight reduction in the initial specific binding (82% and 86%, respectively). After 3 days at 8"C, the control specific binding was reduced to 41% of the initial level. The stability of the hormone binding was improved by 25 mM DTE (74% of control initial binding), 10 mM citrate (6O%), 10% wl v sucrose (%YO),and 10% vlv glycerol (60%). Molybdate (10 mM) and 5 mM EDTA (29% and 23%, respectively) apparently hasten the decay of the receptor. The long-term effect of those compounds, which had proved beneficial in the preliminary experiment, was then tested (Fig. 12). Sucrose (10% wlv) improves the stability of the unoccupied receptor for 3 days, and then the specific binding capacity declines dramatically. This is probably due to microbial contamination, since the addition of sodium azide extends the life of the receptor (50% of the activity remaining after - 5 days). Addition of citrate and DTE, as well as sucrose and sodium azide, improves the stability of the receptor still further (50% of activity remaining after 8.5 days). If the receptor preparation is frozen in the presence of sucrose (10% wlv), citrate (10 mM), and DTE (25 mM) at -20°C or -79"C, the specific binding capacity is totally stable for at least 8 weeks. The receptor shows the same S- - Ecdysteroid Receptors i 311 i STORAGE TIME AT 8% [DAYS] Fig. 12. Stability of the Bll ecdysteroid receptor at 8OC in the absence of hormone. Aliquots (100 PI) of the cell extract were incubated at B0C with 50 pI of solutions containing the required additives in 10 mM HEPES buffer (pH 8). At the required time points, 50 pl of 36 nM [3H]pon A solution (+200-fold excess of unlabelled pon A) were added, and the tubes were incubated for a further 2 h at 2 5 T . Specifically bound hormone was then determined with + sucrose DCC assay B. Control ( + buffer), 0 ; + sucrose (final concentration, 10% w/v), (10%) + NaN, (0.002%, w/v), A; + sucrose (10%) + NaN3 + citrate (10 mM) + DTE (25 mM), *; 0. value after being frozen for 4 weeks at -20°C as the receptor in freshly prepared extract (data not shown). Effect of NaCl on the Stability of the Hormone-Receptor Complex The receptor preparation was prelabelled with 13H]pon A (9 nM 200fold excess of unlabelled pon A) for 2 h at 25°C. Aliquots were then brought to the required temperature ( l ° C or 17°C)and a concentrated NaCl solution was added to give a final NaCl concentration of 0.5 M. The specifically bound label was determined at fixed time points with charcoal assay B. In the presence of 0.5 M NaC1, the hormone-receptor complex is unstable in a temperature-dependent manner. At 1°C the half-life of the H-R complex is approximately 80 min, while at 17°C the half-life is approximately 11 min (data not shown). DISCUSSION The data which are presently available concerning ecdysteroid receptors in insects are derived from four systems: Drosophila salivary glands , Drosophilu imaginal discs , Drosophilu embryos [MI, and Drosuphilu &-cells [l], which, although of uncertain cell typel are believed to be imaginal disc cells . The characterisation of the receptor species from &-cells and imaginal discs, although far from complete, shows many similarities in their properties [2-61. This is perhaps not surprising if they really are of the same cell type. It is therefore of great interest to extend these studies to other cell types of Drosophilu in order to determine if the various target tissues have identical ecdysteroid receptors, 312 Dinan The tumorous blood cell line used in this study is an excellent model system for answering this question and many others concerning the mode of action of ecdysteroids. In addition to being of defined origin [S], the cell line shows an interesting and unusual response to ecdysteroids. The phagocytotic response induced by ecdysteroids [17-191 is similar to the role of haemocytes during metamorphosis in Drosophila [20,21]. The histolysis of larval tissues occurring at the end of dipteran larval life has stimulated the interest of many research workers [22-241, and there is evidence that this process is induced by ecdysteroids [23,25]. The BII and BIII cell lines offer simpler systems for the study of the induction of phagocytosis in haemocytes. In the mutants from which the cell lines are derived, the tumorous blood cells not only phagocytose dying larval tissues, but also larval tissues and imaginal discs, which should survive metamorphosis [S]. Consequently, these cell lines are also important for the study of neoplastic growth. The [3H]pon A used in this study deserves some comment. It was produced by the reductive tritiation of a probable mixture of 24- and 26-dehydro25-deoxy-20-hydroxyecdysone,yielding a product with a specific radioactivity of 180 Cilmmol. This is equivalent to -6 tritium atoms per molecule of ponasterone A. Localisation studies show that effectively all the tritium is located in the side-chain. Since this region of the molecule is structurally important for biological activity , it is perhaps not surprising that the very 'hot' pon A is not recognised by the BII cells in the same way as unlabelled pon A. This isotope effect causes several methodological problems, since standard receptor studies are based on the assumption that labelled and unlabelled ligands are identical with regard to receptor affinity and binding [27l. Radioactive steroids of high specific radioactivity can be expected to be unstable. Hptlc analysis of the radiochemical purity of the [3H]pon A (Fig. 2A) erroneously indicates that there is no degradation, even after 2 years. However, reverse-phase HPLC separation of the same preparation (Fig. 2B) reveals that extensive degradation (-50%) has in fact occurred during this period. Consequently, it is not sufficient to determine the radiochemcial purity by tlc alone. A half-life of -2 years was to be expected for the I3H]pon A since each molecule contains an average of six tritium atoms (tI,* for tritium = 12.1 years). BII cells do not metabolise any of the three ecdysteroids tested and cannot convert ecdysone to 20-hydroxyecdysone. This is in agreement with studies on ecdysteroid metabolism, which have shown that insect haemolymph is not an important metabolic site for ecdysteroids. The specific uptake of pon A by intact BII cells is saturable (Fig. 3). Scatchard plots of this data indicated that the cells do not recognise the labelled and unlabelled pon A equivalently (Fig. 3A). A Scatchard plot obtained solely with [3H]p~nA shows this to be the case (Fig. 3C) and also reveals the finer structure of the binding curve for [3H]pon A. One plausible interpretation of this curve is that it reveals the presence of two specific binding sites, type I being of very high affinity (KD for [3H]pon A = 0.3 nM) with approximately 650 sites per cell, and type I1 being of high affinity (KD for [3H]pon A = 2 nM) with approximately 1,200 sites per cell. However, Ecdysteroid Receptors 313 proof of this interpretation requires further experimentation. Multiple sites for a steroid hormone have previously been described for the oestrogen receptors in rat uterus [28, and references cited therein]. The association of [3H]pon A with BII cells is second order. The association rate constant for [3H]pon A was determined to be 2.6 x 1O7M-'rnin-' at 21°C and 9.6 x 105M-lmin-' at 1°C. Yund et a1  have determined k, for pon A with imaginal discs to be 1.2 x 107M -'min-' at 25°C and 1.4 x 106M-'min-' at 0°C to 4°C. Beckers et a1  have determined the k, for K, cells to be 3 X 107M-'min-' at 20°C. These values are in very good agreement with those determined in the present work. The dissociation kinetics of [3H]pon A show two components (Fig. 5): a rapid dissociation of nonspecifically bound [3H]pon A, which is complete by 25 min at 22"C, followed by a slower dissociation of the specifically bound [3H]pon A with a half-life of 145 min. The dissociation rate constant for this slower component is 4 x 10-3/min. This compares with values of 5.8 x lop3/ min for K, cells  and 3.6 x 10-2/min for imaginal discs . The values for imaginal discs and K, cells were obtained with a r3H]pon A (122 Cilmmol), which apparently has the same biological activity as unlabelled pon A . The lower kd obtained with BII cells might be due to the altered affinity of the [3H]ponA, or it might reflect a difference between the previously studied ecdysteroid receptors and those of BII cells. A value of 0.15 nM for KD is obtained from the quotient of kd and k,, which is in good agreement with the value obtained from equilibrium studies (0.3 nM). From the competition studies (Fig. 6), it is possible to calculate the approximate KD values 1291 for unlabelled pon A (2 nM), 20-hydroxyecdyand ,ecdysone (30 pM). sone (200 a) The KD for unlabelled pon A is similar to that determined for labelled and unlabelled pon A in previous studies [1,2]. Therefore, it would appear that the higher affinity of the BII receptor for the [3H]pon A used in this study, in comparison with that obtained in previous studies, is not due to a difference in the receptor per se, but to the altered stereostructure of the hormone caused by the high degree of tritiation in the side-chain, which fortuitously results in a closer fit of the hormone in the binding site. Therefore, the uptake and binding of [3H]pon A by intact BII cells show three classical attibutes which indicate the presence of ecdysteroid receptors in these cells: specificity, high affinity, and saturability. As a prerequistite to the purification of the ecdysteroid receptor, it is necessary to obtain a cell-free extract containing binding activity and to characterise this binding activity as fully as possible in order to avoid artefactual results during the purification. Additionally, it is important to determine conditions under which the receptor is as stable as possible, both in the presence and absence of hormone. It proved difficult to lyse BII cells homogeneously in hypotonic buffer, and, therefore, a cell-free extract was prepared by sonification. This extract binds [3H]pon A specifically as shown by both DCC assay and chromatography on Sephadex G-25. A good yield of receptor was obtained from the cells if the cells were sonified with one 15-s burst at 40 W. Approximately 60% of the specific binding activity found with whole cells is recovered in the supernatant after centrifugation at 20,OOOg. The optimum pH for extraction 314 Dinan and binding to the receptor is 8.2. This compares with a broad optimum pH of 7-9, which has been found for the ecdysteroid receptor in the crayfish Astucus Zeptodactylus . The presence of divalent metal ions results in reduced binding to the receptor. Calcium and magnesium ions cause 85% inhibition of the control binding at 50 mM. Magnesium is not so inhibitory (50% inhibition at 100 mM). Monovalent cations (Na+ and K+)show a reproducible stimulation of specific binding at low concentrations ( <75 mM) if the preparation is prepared in TrislHCl buffer (75mM, pH 8). If, however, the cells are sonified in HEPES buffer (10 mM, pH 8), no such stimulation is seen at low NaCl concentrations; all concentrations of NaCl(25-250 mM) were inhibitory, with 50% inhibition at 200 mM (Dinan, unpublished observation). The reason for this difference between the two buffer systems is unknown. The H-R complex is also unstable in the presence of high salt concentrations. It decays rapidly in the presence of 0.5 M NaCl in a temperature dependent fashion. Mammalian steroid hormone receptors have also been found to be unstable in the presence of high salt concentrations [31,32], a process which can be hindered by the addition of glycerol . The sonification preparation shows a saturable specific binding of [3H]pon A (Fig. 8A). If these results are expressed according to Scatchard (Fig. 8B), a curve is obtained, which is similar to that obtained with whole cells (Fig. 3C). This curve can be resolved into two components: a very high affinity specific component (KD = 10-loM; 10 fmollmg protein) and a high affinity specific component (KD = 1nM; 30 fmollmg protein). Specifically bound [3H]pon A is macromolecularly bound, as shown by chromatography of the preincubated cell extract on Sephadex G-25 (Fig. 9). The radioactivity eluting in the macromolecular fraction is totally compatible with 100-fold excess of unlabelled pon A. The nature of the macromolecular binding component was investigated using several digestive enzymes (Table 1).The most effective enzymes were the proteases, pronase and trypsin, indicating that pon A binds to a protein. The heat instability of the binding and its susceptibility to NEM also indicate this. The effect of NEM also suggests that a sulphydryl group is important for binding activity. Both DNase and RNase also slightly reduce the amount of [3H]pon A specifically bound. In the case of the DNase, this can probably be attributed to the presence of contaminating protease activity (detected by the "azocasein method"), which is not inhibited by aprotinin (Dinan, unpublished observation). The RNase preparation does not show any protease activity in the azocasein test, but azocasein may not be a suitable substrate and the test is very insensitive. Triton X-100 and Nonidet NP-40, when present at concentrations as low as 0.1% vlv, totally prevented the adsorption of unbound [3H]pon A to charcoal. Consequently, it was necessary to separate the free and bound hormone on Sephadex G-25 in order to test the effect of these compounds on binding. When added at the same time as the labelled pon A, both detergents prevent all specific macromolecular binding, When the receptor preparation was equilibrated with C3H]pon A and then NP-40 (0.8%) was added just before application to the Sephadex G-25 column, the amount of Ecdysteroid Receptors 315 [3H]pon A macromolecularly bound was identical with the control without NP-40 (24% of added label). Therefore, these detergents cannot be used for the extraction of naive ecdysteroid receptors, but could be used to lyse cells which have already been incubated with hormone. Triton X-100 has been shown to increase the dissociation of mammalian steroid hormones from their receptors . The ecdysteroid-receptor complex from BII cells sediments as one peak on sucrose density gradients with an S-value of approximately 6S, which is similar to the value obtained for the ecdysteroid receptors from Dvosophilu embryos and imaginal discs under low salt conditions  and for that obtained for the ecdysteroid receptor from freshly harvested K, cells . A similar value (6.6s) was obtained if an unlabelled BII cell extract was separated by sucrose gradient centrifugation, fractionated, and the specific binding determined in the individual fractions (data not shown). In common with many other steroid hormone receptors, the ecdysteroid receptor is more stable in crude preparations in the presence of ligand than in its absence (Fig. 11).The half-life for the unloaded receptor at 8°C in the presence of aprotinin (which was added as a protease inhibitor) is 3 days. Aprotinin extends the life of the H-R complex at room temperature (Dinan, unpublished observation). The H-R complex is stable for at least 3 days at 8°C but rapidly decays at 25°C. The stability of the unloaded receptor at 8°C is improved by the presence of thiol reagents, sucrose, glycerol, or citrate. The first three compounds have been shown to be beneficial for the stability of many steroid receptors [35-381. Citrate has been shown to improve the stability of the rat hepatic glucocorticoid receptor , although the mechanism is not clear. Both molybdate and EDTA have been shown to stabilise certain receptors [31,36,38,40,41]. However, this does not appear to be the case with the ecdysteroid receptor from BII cells since they both accelerate the decay of binding activity. A combination of the additives 10% sucrose, 0.02% NaN3, 10 mh4 citrate, and 25 mh4 DTE considerably improves the stability of the ecdysteroid receptor at 8°C (Fig. 12). When frozen at -20°C in the presence of these additives, the preparation is totally stable for a considerable length of time. If the frozen receptor preparation (4weeks old) is thawed, incubated with [3H]ponA (2 h at 25°C) and then separated by sucrose gradient centrifugation, a single peak at 6s is obtained (Dinan, unpublished observation). Thus, it has been possible to partially characterise the high affinity binding proteins for [3H]pon A in a cell-free extract of a tumorous blood cell line of Drosophilu, which responds to ecdysteroid treatment. Many interesting questions remain to be investigated. Particularly important is the location of the receptor within the cell since both imaginal discs  and K, cells  possess unoccupied nuclear as well as cytosolic receptors. LITERATURE CITED 1. Mar6y P, Dennis R, Beckers C, Sage BA, O’Connor JD: Demonstration of an ecdysteroid receptor in a cultured cell line of Drosophilu melnnoguster. Proc Natl Acad Sci USA 75, 6035 (1978). 316 Dinan 2. Yund MA, King DS, Fristrom JW: Ecdysteroid receptors in imaginal discs of Drosophila melnnoguster. 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Pavlik EJ, Rutledge S: Estrogen-binding properties of cytoplasmic and nuclear estrogen receptors in the presence of Triton X-100. J Steroid Biochem 13, 1433 (1980). 34. Osterbur DL, Yund MA: Ecdysteroid binding activity in embryos of Drosophila melanogaster. J Cell Biochem 20, 277 (1982). 35. Schmid W, Grote H, Sekeris CE: Stabilization and characterization of the dexamethasonebinding proteins in rat liver cytosol. Mol Cell Endocrinol 5, 223 (1976). 36. Rees AM, Bell PA: The involvement of receptor sulphydryl groups in the binding of steroids to the cytoplasmic glucocorticoid receptor from rat thymus. Biochim Biophys Acta 411, 121 (1975). 37. Schaumberg BP: Investigations on the glucocorticoid-binding protein from rat thymocytes. 11: Stability, kinetics and specificity of binding of steroids. Biochim Biophys Acta 261, 219 (1972). 38. McBlain WA, Shyamala G : Inactivation of mammary cytoplasmic glucocorticoid receptors under cell-free conditions. J Biol Chem 255, 3884 (1980). 39. Hubbard J, Kalimi M: Alteration of hepatic glucocorticoid receptor stability and nuclear binding in nitro by citrate. Biochem J 210, 259 (1983). 40. Chen TJ, MacDonald RG, Leavitt WW: Uterine progesterone receptor: Stabilization and physicochemical alterations produced by sodium molybdate. Biochemistry 20, 3504 (1981). 41. Narly A: Molybdate effect on the glucocorticoid receptor in cell-free systems and intact lymphocytes. Biochim Biophys Acta 756, 328 (1983).