Magnetic DNA affinity purification of ecdysteroid receptor.

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity.

High-resolution gel filtration of the ecdysteroid receptor-DNA complex--an alternative to the electrophoretic mobility shift assay.

The mobility shift assay is a well established method for proving binding of protein to DNA. However, this method depends on the stability of the protein-DNA complex during the electrophoretic process. Ecdysteroid receptor shows a strong tendency to aggregate under low-salt conditions of electrophoresis to a non DNA-binding form.

Characterization of a specific ecdysteroid receptor-DNA complex reveals common properties for invertebrate and vertebrate hormone-receptor/DNA interactions.

A gel-filtration assay has been developed with which the specific interaction between Drosophila melanogaster ecdysteroid receptor and the 20-hydroxy-ecdysone responsive element of the hsp27 gene promoter region was characterized in terms of complex formation, saturation of DNA binding and the apparent molecular mass of the complex. The hsp27 DNA-binding sequence for ecdysteroid receptor in vitro was delimited by footprinting and mutational analysis. The combined results show that ecdysteroid receptor binds as a dimer to an imperfect palindromic sequence (GGTTCAATGCACT) closely resembling the structures of the different vertebrate steroid-hormone-responsive elements reported so far.

Pyridoxal phosphate inhibits the DNA-binding activity of the ecdysteroid receptor.

The effect of pyridoxal 5'-phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA-cellulose was studied. The binding of hormone-receptor complexes to DNA-cellulose was completely blocked after a 30-min incubation with 3 mM pyridoxal 5'-phosphate at 0-4 degree C. The effect was specific for pyridoxal 5'-phosphate since related compounds (pyridoxal, pyridoxamine 5'-phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal).

Affinity labelling of a partially purified ecdysteroid receptor with a bromoacetylated 20-OH-ecdysone derivative.

The novel bromoacetyl ecdysteroid IV, (20R,22R)-2 beta,3 beta,14 alpha,20,22,25 xi-hexahydroxy-26-(3- bromoacetoxypropyl)-5 beta-cholest-7-en-6-one, BAEIV, has been synthesized by extending the side chain on C26 of 20-OH-ecdysone. BAEIV meets all the requirements for an affinity-labelling reagent. It reacts with the partially purified ecdysteroid receptors of Drosophila melanogaster rapidly and almost quantitatively.