Ecdysteroid receptors of the blowfly Calliphora vicina: partial purification and characterization of ecdysteroid binding.
A macromolecule with high affinity for the ecdysteroid analogue ponasterone A was isolated from nuclei of larvae of the blowfly Calliphora vicina. The ecdysteroid-binding molecule revealed characteristics of the moulting hormone receptor. It was sensitive towards protease but not towards nucleases. The nuclear protein had a limited binding capacity (0.2 pmol ponasterone A/mg protein), showed hormone analogue specificity and high affinity for ecdysteroids. Enzyme activities were present in the nuclear extract that metabolized ecdysteroids and thereby interfered with the binding assay. After their removal by DEAE-cellulose chromatography the ecdysteroid receptor preparation was stable at 20 degrees C for hours. This allowed a reliable determination of dissociation constants at equilibrium conditions. The hormone receptor complex had a KD of 1 nM, 30 nM, and 2000 nM with ponasterone A, 20-hydroxyecdysone, and ecdysone, respectively. The apparent molecular mass of the ecdysteroid receptor was 105,000 as determined by gel filtration.
Lehmann
M
M
Physiologisch-Chemisches Institut, Philipps-Universität, Marburg, F.R.G.
Koolman
J
J
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000648:ultrastructure; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004175:Diptera / Q000166:cytology* / Q000196:embryology / Q000648:ultrastructure; D026461:Ecdysteroids; D007447:Invertebrate Hormones / Q000302:isolation & purification* / Q000378:metabolism / Q000493:pharmacokinetics; D007814:Larva / Q000166:cytology / Q000648:ultrastructure; D011863:Radioimmunoassay; D011987:Receptors, Steroid / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism / Q000493:pharmacokinetics
D026461:Ecdysteroids; D007447:Invertebrate Hormones; D011987:Receptors, Steroid; C034808:ecdysteroid receptor