Early loss of the tyrosyl radical in ribonucleotide reductase of adenocarcinoma cells producing nitric oxide.
Nitric oxide (NO) has been previously shown to inhibit crude preparations of ribonucleotide reductase, a key enzyme in DNA synthesis, and to destroy the essential tyrosyl free radical in pure recombinant R2 subunit of the enzyme. In R2-overexpressing TA3 cells, a decrease in the tyrosyl radical was observed by whole-cell EPR spectroscopy, as soon as 4 h after NO synthase induction by immunological stimuli. Complete loss of the tyrosyl EPR signal occurred after 7 h in cells cultured at a high density. Disappearance of the tyrosyl radical was prevented by N omega-nitro-L-arginine, a specific inhibitor of NO synthesis, and by oxyhemoglobin, which reacts rapidly with NO. It was reproduced by S-nitrosoglutathione, a NO-releasing molecule. Stable end products of NO synthase metabolism did not affect the radical. Immunoblot analysis of the R2 subunit indicated that expression of the protein was not influenced by NO synthase activity. These results establish that NO, or a labile product of NO synthase, induces the disappearance of the R2-centered tyrosyl radical. Since the radical is necessary for ribonucleotide reductase activity, its destruction by NO would contribute markedly to the antiproliferative action exerted by macrophage-type NO synthase.
Lepoivre
M
M
Unité 1116 Centre National de la Recherche Scientifique, Université Paris-Sud, Orsay, France.
Flaman
J M
JM
Henry
Y
Y
D000230:Adenocarcinoma / Q000201:enzymology*; D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D002956:Citrulline / Q000032:analysis / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004790:Enzyme Induction; D005609:Free Radicals; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D009573:Nitrites / Q000032:analysis / Q000378:metabolism; D019335:Nitroarginine; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine
D005609:Free Radicals; D009573:Nitrites; D019335:Nitroarginine; D002956:Citrulline; D009569:Nitric Oxide; D014443:Tyrosine; D001120:Arginine; D019001:Nitric Oxide Synthase; D012264:Ribonucleotide Reductases; D000594:Amino Acid Oxidoreductases