Characterization of cytoplasmic ecdysteroid receptors in the hypodermis of the crayfish, Orconectes limosus.

[3H]Ecdysone, [3H]20-OH-ecdysone and [3H]ponasterone A were specifically bound by the 120 000 g supernatant of the homogenate from crayfish hypodermis. According to the HPLC test, the bound hormone was not metabolized. Analysis of the cytosol receptor by sucrose-density-gradient centrifugation, gel filtration and enzymatic degradation revealed the protein character of the receptor, a molecular weight of 70 000-80 000 D, and 5S. The binding capacity was rapidly lost after treatment with N-ethyl-maleimide or heating for 10 min at 60 degrees C. Optimal conditions for the demonstration of the receptor were incubation at 0-8 degrees C for 30 min and a salt concentration of 120 mM. A sequence of moulting hormones, and steroid hormones without moulting hormone activity were tested for ligand specificities. Estimation KD values yielded 3-6 x 10(-11) M for ponasterone A, 2-3 x 10(-9) M for 20-OH-ecdysone and 2 x 10(-8) M for ecdysone.