Characterization of nuclear ecdysteroid receptor from crayfish integument

The existence of nuclear ecdysteroid receptors in isolated nuclei from crayfish integument was demonstrated. Nuclei were prepared from crayfish hypodermis by use of a modified Chauveau-method and exposed to [3H]-ecdysteroids. The nuclear hormone receptor complex sedimented at about 5 S and the available binding sites had a high affinity to [3H]-ecdysteroids. Treatment of the nuclei with pronase and DNase completed or partially reduced nuclear bound ecdysone, whereas RNase was without any effect. Competition of nuclear bound ecdysone by various steroids confirmed ponasterone A and kaladasterone as the best competitors followed by 20-OH-ecdysone and ecdysone, whereas compounds like poststerone, "triol" or hydrocortisone did not compete. If [3H]-20-OH-ecdysone was used as the radio-ligand, 20-OH-ecdysone was a better competitor than ecdysone indicating that there may be two different binding sites for ecdysone and 20-OH-ecdysone; this was further substantiated by the finding of two different number of binding sites for these steroids.