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). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)-2 beta,3 beta, 14 alpha,20,22-pentahydroxy-5 beta-cholest-7-en-6-one ([3H]ponasterone A)-receptor complexes. The loss of DNA-binding activity caused by pyridoxal 5'-phosphate is accompanied by changes in the molecular properties of [3H]ponasterone-A-receptor complexes. A shift of [3H]ponasterone-A binding was observed from the 8.0-8.5 S to the 4.5-5.0 S region, when [3H]ponasterone-A-receptor complexes were exposed to pyridoxal 5'-phosphate during sucrose-gradient centrifugation. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed. Probably, pyridoxal 5'-phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA-binding site. The hormone-receptor complexes obtained after removal of pyridoxal 5'-phosphate had the same affinity for DNA-cellulose as 'native' complexes. DNA-cellulose-bound [3H]ponasterone-A complexes were efficiently eluted from DNA-cellulose with pyridoxal 5'-phosphate in 0.1 M KCl resulting in a 104-fold purification of the ecdysteroid receptor. The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.
Lehrstuhl für Biochemie, Ruhr-Universität Bochum, Federal Republic of Germany.
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Ozyhar
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Kiltz
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D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002845:Chromatography / Q000379:methods; D004247:DNA / Q000378:metabolism*; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology*; D011987:Receptors, Steroid / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014316:Tritium
C033190:DNA-cellulose; D011987:Receptors, Steroid; C034808:ecdysteroid receptor; D014316:Tritium; D004441:Ecdysterone; D011732:Pyridoxal Phosphate; C008999:ponasterone A; D002482:Cellulose; D004247:DNA