DNA dependence and inhibition by novobiocin and coumermycin of the nucleolar adenosine triphosphatase (ATPase) of human fibroblasts.
We have recently demonstrated by electron microscopic cytochemical methods that unfixed human fibroblasts exhibit intense MG2+ dependent adenosine triphosphatase (nATPase) activity in circumscribed areas of the cell nucleoli. The nATPase was specific for ATP and dATP and was inhibited by other ribonucleoside triphosphates. Its intranucleolar localization relative to nucleolar chromatin, and segregation into nucleolar zones after actinomycin treatment of the cells, suggested that the reaction took place in fibrillar centers. This ATPase has now been further characterized by electron microscopic cytochemistry. It was determined that short fixation permitted retention of most of the ATPase activity, and that the enzyme was active at high ionic strength (up to 400 mM KCl), but that the enzyme activity was very sensitive to elevated temperatures. DNA dependence of the enzyme was shown by inhibition of the reaction by DNase pretreatment in parallel with the removal of DNA from the cell, while pretreatment with RNase had no significant effect. The nATPase activity was also selectively inhibited by treatment of the cells with antagonists of the B subunit of DNA gyrase, novobiocin, and coumermycin, but not by nalidixic or oxolinic acids, which interfere with the A subunit of gyrase. Inhibitors of RNA synthesis, actinomycin D and aminonucleoside of puromycin, potentiate rather than inhibit nATPase reaction. The results suggest that nATPase functions to alter the degree of supercoiling or catenation of nucleolar organizer DNA, and is in reality a DNA topoisomerase that hydrolyzes ATP during its action.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D049933:Aminocoumarins; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002466:Cell Nucleolus / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D003374:Coumarins / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000196:embryology; D008854:Microscopy, Electron; D009675:Novobiocin / Q000494:pharmacology*; D009994:Osmolar Concentration; D014158:Transcription, Genetic / Q000187:drug effects
D049933:Aminocoumarins; D000900:Anti-Bacterial Agents; D003374:Coumarins; D009675:Novobiocin; D003609:Dactinomycin; D004247:DNA; D000251:Adenosine Triphosphatases; C004628:coumermycin