The effects of 1,10-phenanthroline on the binding of activated rat hepatic glucocorticoid-receptor complexes to deoxyribonucleic acid-cellulose.
1,10-Phenanthroline, a metal ion chelator, inhibits the binding of previously activated (25 C for 30 min) rat hepatic [3H]triamcinolone acetonide (3H-labeled 9-fluoro-11 beta, 21-dihydroxy-16 alpha, 17-1-[1-metylethylidenebis(oxy)]pregna-1,4-diene-3,20-dione ([3H]TA)-receptor complexes to DNA-cellulose. The observed inhibition increases as the temperature of the preincubation with chelator is increased from 0 to 25 C. Fifty percent of the maximal inhibition (greater than 90%) detected at 25 C is achieved with 1 mM 1,10-phenanthroline. The observed inhibition is not the consequence of DNA degradation by 1,10-phenanthroline-Cu2+ complexes, since preincubation of activated cytosol with neocuproine (2,9-dimethyl-1,10-phenanthroline), a potent Cu2+ chelator, fails to block the subsequent inhibition of DNA-cellulose binding by 1,10-phenanthroline. The failure of other chelators which complex siilar metal ions (alpha, alpha'-dipyridyl,8-hydroxyquinoline, 2,2',2"-tripyridine, EDTA, EGTA, and Na azide) to inhibit DNA-cellulose binding suggests that the effectiveness of 1,10-phenanthroline does not result from removal of a required free metal ion(s) but, rather, from a specific interaction with a metal ion(s) which may be located within the activated receptor protein. The observed inhibition is dependent on the metal chelating properties of 1,10-phenanthroline, since preincubation with several divalent metal cations (Zn2+, Co2+, and Ni2+) which are known to be chelated by this compound block its subsequent inhibitory effect. Ferroin (1,10-phenanthroline-ferrous sulfate complex) and 1,7-phenanthroline (nonchelating isomer) also fail to inhibit DNA-cellulose binding. The inhibition mediated by 1,10-phenanthroline persists after gel filtration, suggesting that 1,10-phenanthroline associated with a macromolecule is the effective form of the inhibitor, rather than free 1,10-phenanthroline. Finally, 1,10-phenanthroline appears to interact directly with activated [3H]TA-receptor complexes, since it alters their net charge and results in their elution from DEAE-cellulose at a salt concentration characteristic of unactivated complexes. Collectively, the data suggest that the activated [3H]TA-receptor complex is a metalloprotein and that the metal ion(s) may be associated directly with the DNA-binding site or may regulate this site indirectly through an allostreic mechanism.
D000315:Adrenalectomy; D000818:Animals; D002413:Cations, Divalent; D002482:Cellulose; D002614:Chelating Agents / Q000494:pharmacology; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D004247:DNA; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010618:Phenanthrolines / Q000494:pharmacology*; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014222:Triamcinolone Acetonide / Q000378:metabolism
D002413:Cations, Divalent; D002614:Chelating Agents; D010618:Phenanthrolines; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D002482:Cellulose; D004247:DNA; D014222:Triamcinolone Acetonide; C025205:1,10-phenanthroline