The Effects of 1,10-Phenanthroline on the Binding of Activated Rat Hepatic Glucocorticoid-Receptor Complexes to Deoxyribonucleic Acid-Cellulose*

Publisher Website
Google Scholar
Abstract
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 llβ,-dihydroxy-16α,17-l-[l-methylethylidenebis(oxy)]pregnal, 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 (>90%) detected at 25 C is achieved with 1 HIM 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-l,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 similar metal ions (α,α′-dipyridyl, 8-hydroxyquinoline, 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 allosteric mechanism.