title: Tyrosine nitration affects thymidylate synthase properties.
abstract: Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.
authors.affiliation: Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Dąbrowska-Maś
authors.forename: Tomasz
authors.initials: T
authors.lastname: Frączyk
authors.forename: Tomasz
authors.initials: T
authors.lastname: Ruman
authors.forename: Karolina
authors.initials: K
authors.lastname: Radziszewska
authors.forename: Piotr
authors.initials: P
authors.lastname: Wilk
authors.forename: Joanna
authors.initials: J
authors.lastname: Cieśla
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Zieliński
authors.forename: Agata
authors.initials: A
authors.lastname: Jurkiewicz
authors.forename: Barbara
authors.initials: B
authors.lastname: Gołos
authors.forename: Patrycja
authors.initials: P
authors.lastname: Wińska
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Wałajtys-Rode
authors.forename: Andrzej
authors.initials: A
authors.lastname: Leś
authors.forename: Joanna
authors.initials: J
authors.lastname: Nizioł
authors.forename: Adam
authors.initials: A
authors.lastname: Jarmuła
authors.forename: Piotr
authors.initials: P
authors.lastname: Stefanowicz
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Szewczuk
authors.forename: Wojciech
authors.initials: W
authors.lastname: Rode
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology; D002417:Cattle; D045744:Cell Line, Tumor; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014443:Tyrosine; D013940:Thymidylate Synthase