Superoxide dismutase and catalase are required to detect (.-)NO from both coupled and uncoupled neuronal no synthase.

Despite numerous approaches to measuring nitric oxide ((.-)NO) formation from purified NO synthase (NOS), it is still not clear whether (.-)NO is a direct or indirect product of the NO synthase reaction. The direct detection of catalytically formed (.-)NO is complicated by side reactions with reactive oxide species like H(2)O(2) and superoxide. The aim of the present study was therefore to reinvestigate these reactions both electrochemically and by chemiluminescence detection with particular emphasis on the requirement for cofactors and their interference with (.-)NO detection. Flavins were found to generate large amounts of H(2)O(2) and were therefore excluded from subsequent incubations. Under conditions of both coupled and uncoupled catalysis, SOD was absolutely required to detect (.-)NO from NOS. H(2)O(2) formation took place also in the presence of SOD and gave a smaller yet significant interfering signal. Similar data were obtained when the proposed intermediate N(omega)-hydroxy-l-arginine was utilized as substrate. In conclusion, standard Clark-type ()NO electrodes are cross-sensitive to H(2)O(2) and therefore both SOD and catalase are absolutely required to specifically detect (.-)NO from NOS.

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide.

The underlying mechanisms regulating the activity of the family of homodimeric nitric oxide synthases (NOSs) and, in particular, the requirement for (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H(4)Bip on neuronal NOS (NOS-I) during the conversion of substrate, L-arginine, into L-citrulline and nitric oxide. Indeed, in kinetic studies dual allosteric interactions between L-arginine and H(4)Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H(4)Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydrooxazolo[1, 2-f]-pteridine (PHS-32), caused an L-arginine-dependent increase in the haem Soret band, indicating an increase in substrate binding to recombinant human NOS-I. Conversely, L-arginine was observed to increase in a concentration-dependent manner H(4)Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H(4)Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H(4)Bip, dimeric recombinant human NOS-I dissociated into inactive monomers. Monomerization was related to the uncoupling of reductive oxygen activation, because it was inhibited by both superoxide dismutase and the inhibitor N(omega)-nitro-L-arginine. Importantly, H(4)Bip was found to react chemically with superoxide (O(2)(-.)) and enzyme-bound H(4)Bip was consumed under O(2)(-.)-generating conditions in the absence of substrate. These results suggest that H(4)Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O(2)(-.).

3-(2,2,2-Trimethylhydrazinium)propionate (THP)--a novel gamma-butyrobetaine hydroxylase inhibitor with cardioprotective properties.

A protein fraction containing gamma-butyrobetaine hydroxylase (sp.act. 1.54 mU/mg) was isolated from the rat liver by differential precipitation with ammonium sulphate. 3-(2,2,2-Trimethylhydrazinium)propionate (THP), a noncompetitive enzyme inhibitor, when administered orally to rats for 10 days (150 mg/kg) elicited a reduction in myocardial free carnitine and long-chain acyl carnitine content by 63.7 and 74.3%, respectively. This reduction in free carnitine concentration causes a suppression of the free fatty acid oxidation, as measured by the production of 14CO2 and ketone bodies. The inhibition of fatty acid oxidation is particularly manifest when their metabolism is stimulated by feeding a fat-rich diet to the animals or in fasting rats. The inhibition of fatty acid metabolism at the stage of activation (acyl carnitine formation) can account for the cardioprotective effect of THP, which is assessed by its ability to prevent a decrease in ATP level and myocardial energy charge as well as to prevent a rise in creatine phosphokinase and lactic dehydrogenase (myocardium-specific isozyme) activity in rat blood serum in response to isoproterenol and epinephrine. Regulation of the carnitine-dependent fatty acid metabolism in ischaemia is a pathogenetically justified approach to pharmacological treatment of ischaemic myocardium. In its biochemical mechanism, THP significally distinguishes itself from other known inhibitors of fatty acid oxidation.