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REGULATION OF NITRIC OXIDE/SUPEROXIDE FORMATION BY TETRAHYDROBIOPTERIN

mMaver, Andrea Leber Burkhard Klijsch and Kurt Schmidt

Institut fiir Pharmakologie und Toxikologie, Karl-Franzens- Universitit Graz, Universititsplatz 2, A-8010 Graz, Austria

Tetrahydrobiopterin (IQB) is an essential nitric oxide syntbase (NOS) cofactor. Besides having well established allosteric effects on the NOS protein (dimer stabilization, low spin-to-high spin shift of the heme), lQl3 appears to par&i

P ate in L-arginine oxida-

tion through novel l-electron chemistry . In tbe absence of HJB some NOS isoforms exhibit substantial uncoupled NADPH ox- idase activity leading to superoxide (02’7 production even if saturated with L-arginine. On the other hand, autooxidation of H&t leads non-enzymatic w- generation and, again, inactivation of NO. Thus, oxidative depletion of cellular H4B results in both enzymatic and non-enzymatic formation of 02’-. This may have important biological consequences because of the rapid reaction of NO with m- yielding the potent cytotoxin peroxynitrite. The so far best characterized pathology which has been suggested to be associated with increased m- formation caused by m depletion is endothelial dysfunction in artberosclerosis and other vascular diseases2. This view is supported by our new data obtained with recombinant human eNOS, which turned out to be a highly active NADPH oxidase at suboptimal H4B levels3.

‘Bet, N., Gorren, A.C.F., Mayer, B. & Lange, R., J. Biol. Chem. 273, 13502-8, 1998. *Cosentino, F. & Liischer, T.F. Eur. Heart J. 19 (Suppl. G), G3-G8. 3Leber, A., Hemmens, B., Kl&sch, B., Goessler! W., Raber, G., Mayer, B. & Schmidt, K. (submitted for publication)

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THERMODYNAMIC LINKAGE RELATIONSHIPS IN S-NITROSOHEMOGLOBIN: OXYGEN BINDING AND VASOACTIVITY

$%%?S, McMahon Anne E. Stone, Ioseph Bonauentura, David 1, Sin el Stamler. Duke LIniversih/ Medical Center, Durham, NC X%0.

The formation of S-nitrosohemoglobin (SNO-Hb) in vitro and in vivo is linked to the oxygen-induced allosteric transition [from the T (deoxy) to the R (oxy) structure] that governs Hb function. These studies predict that covalent modification of the Cysf193 residue by S-nitrosylation should increase oxygen affinity, and indeed recent reports from this and other laboratories show this to be true. Our data also predict that NO group release is favored under deoxy conditions. Surprisingly, though, recent results of Pate1 et al (JBC 274:15487-15492,1999) were interpreted to suggest that transnitrosation between SNO-Hb and GSH (forming GSNO) is slower in the deoxy conformation. This interpretation runs contrary to the most fundamental principles of thermodynamic @linkagei relationships in hemoglobin (Jeffries Wyman of MWC fame). Here we present the results of oxygen-binding and bioassay experiments designed to address this apparent paradox, and show that the new data are consistent with thermodynamic tenets.

EPR EVIDENCE FOR PEROXIDASE-DEPENDENT FORMATION OF A NITRITE-DERIVED OXIDANT

ML MCCORMICK, KJ BESZKA, SL HAZEN, GR BUETT~~R, BE BRITIGAN. VA Medical Center, Univ.of Iowa, Iowa City, IA and Cleveland Clinic Foundation, Cleveland, OH.

Several laboratories have shown that peroxidases are able to both oxidize and nitrate target molecules using NOs- as a substrate. We have investigated the mechanism of oxidation of target proteins by peroxidase/HsOz/NOa- systems using MNP spin trapping of tyrosyl radical (‘TYR) generated on a synthetic oligopeptide target (R) containing 20% tyrosine. Addition of myeloperoxidase, eosinopbil peroxidase, or lactoperoxidase to H202 and NOz‘ in the presence of oligopeptide target molecules led to formation of protein-bound MNPYTYR-R adducts readily detectable by EPR following pronase treatment. No MNPrTYR signal was observed if peroxidase, HsOs, or NOi was omitted. When alternative oligopeptides lacking tyrosine residues were employed, no MNP adducts were detected. EPR spin trapping studies using authentic peroxynitrite (as a potential product of EPOIH20siNOs~ activity) to oxidize tyrosine residues in target proteins are currently underway. We also used the spin trap DMPO to study these reactions. Reaction of DMPO in the presence of eosinophil peroxidase and Hz02 yielded DMPOX, which was greatly enhanced in the presence of NOz- but was also observed with the addition of 500 uM peroxynitrite to DMPO. Our data are consistent with peroxidase/HsOs/NOz~ systems forming a reactive specie capable of oxidizing protein-bound tyrosine to protein-bound tyrosyl radical.

piq

NITRIC OXIDE ENHANCES THE MNSOD-DEPENDENT SUPPRESSION OF PROLIFERATION IN FIBROSARCOMA CELLS.

Kwi-H _Y

e Kim, Ana M. Rodriqua and Kelvin 1. A. Davies. Afbnny Medical College, A bany, NY 12208.

It is believed that the anti-tumoral immune response is in part mediated by the release of NOo from immune cells. In addition, Superoxide dismutase has been shown to enhance the biological activity of NO” and the toxicity of No” generating compounds. For these reasons we tested whether NO” contributes to the reduction in tumorigenicity associated with manganese superoxide dismutase (MnSOD) overexpression. The effects of a variety of No” generating compounds on the proliferative capacity of control and MnSOD overexpressing fibrosarcoma cells were tested. MnSOD enhanced the cytostatic properties of the NO” donors, SNP, SIN-1 and DETANONOate in a dose dependent fashion when compared to control cells. MnSOD has also been shown to catalyze ONOO-dependent nitration reactions. To evaluate the contribution of nitration reactions to the enhanced proliferative inhibition of No” on MnSOD overexpressing cells 3-nitrotyrosine levels were analyzed. Immunoblot analysis of lysates from both control and MnSOD overexpressing cells demonstrated the specific loss of a 130 -150 kD 3-nitrotyroslne imrnunoreactive band in MnSOD overexpressing cells. Thus, a decrease in tyrosine nitration is associated with MnSOD overexpression. The mechanism for the MnSOD-dependent decrease in tyrosine nitration is as yet unclear. Nonetheless, these findings suggest that NOo may play a role in the reversal of tumorigenicity associated with MnSOD overexpression.

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