Nitric oxide synthases (NOS) require NADPH and tetrahydrobiopterin (H4biopterin) to convert L-arginine to L-citrulline. The additional requirement and effects of thiols during purification and activity assays of NOS are unclear; for example, glutathione (GSH) has been reported to stimulate or, in the presence of catalase, to inhibit enzyme activity. We therefore studied the effects of different thiols, thiol reagents, antioxidants, and H4biopterin-regenerating systems on purified porcine cerebellum NOS. GSH in the presence of catalase did not inhibit NOS. In contrast, GSH and, to a lesser degree, several other thiols consistently stimulated total L-arginine turnover up to 4-fold. In the presence of GSH, Vmax of NOS was increased, the usually observed loss of activity during the 15 min assay was less dramatic, and the apparent S0.5 value for H4biopterin decreased. Stabilization of NOS activity by GSH was augmented by protein disulfide isomerase (PDI), indicating that, at least in part, GSH acted by reductive protection of NOS protein thiols. Consistent with this, four different protein thiol reagents abolished NOS activity. In other experiments, specific allosteric binding was excluded as a potential mechanism of GSH regulation of NOS. In addition, GSH may affect NOS kinetics by recycling or preventing the autoxidation of H4biopterin. In support of this, the non-thiol reductant ascorbate and dihydropteridine reductase mimicked the effects of GSH on NOS kinetics, but not on NOS stability. Thus, NOS activity depends on both H4biopterin and the reduced state of essential protein thiols.
[Show abstract][Hide abstract] ABSTRACT: Peroxynitrite (PN) can be formed under mainly pathophysiological conditions from nitric oxide (NO) and superoxide anion and may be responsible for oxidative modifications of biomolecules. Preparations of nitric oxide synthases from porcine cerebellum (nNOS), bovine aortic endothelium (eNOS) and cytokine-treated murine macrophages (iNOS) were inhibited by PN in their ability to transform arginine to citrulline and nitric oxide with IC50 values of 15, 28, and 10 microM, respectively. Glutathione, bovine serum albumin and tyrosine provided varying degrees of protection in the three preparations. Intact endothelial cells, upon exposure to PN, rapidly lost their glutathione content but protein-SH groups and eNOS activity remained largely unaffected. Destruction of the heme-thiolate catalytic site was observed when nNOS was exposed to PN suggesting that the irreversible oxidation of this bond may be the common mechanism of NOS inhibition.
[Show abstract][Hide abstract] ABSTRACT: Indications for the occurrence of nitric oxide synthases in Dictyostelium, Neurospora, Phycomyces and the leguminous plant Mucuna hassjoo as well as a physiological role of nitric oxide in Neurospora crassa are demonstrated. An exogenous nitric oxide donor, sodium nitroprus-side, inhibited light-stimulated conidiation in N. crassa. Specific inhibitors of nitric oxide synthase, like the arginine derivatives NG-nitro-L-arginine (L-NA) and NG-nitro-L-arginine-methyl ester (L-NAME), enhanced conidiation in darkness and in the light, whereas the stereo-isomer D-NAME was inactive. This communication reports to our knowledge the first time the presence of enzymatic activity of nitric oxide synthase in fungi and a higher plant and an effect of nitric oxide in fungal photophysiology.
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