flavin-adenine-dinucleotide and sapropterin

The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.

Topics: Antioxidants; Arginine; Biopterins; Catalysis; Citrulline; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Heme; Hydrogen Peroxide; Isoenzymes; Nitric Oxide; Nitric Oxide 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.

Topics: Arginine; Biopterins; Catalase; Electrochemistry; Electrodes; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Humans; Hydrogen Peroxide; Hydroxylation; Luminescent Measurements; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Superoxide Dismutase

The activity and expression of nitric oxide synthase (NOS) isoforms and protein nitrotyrosine (NT) residues were investigated in whole encephalic mass (WEM) homogenates during the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. EAE stages (0-III) were daily defined by clinical evaluation, and in the end of each stage, WEMs were removed for analysis of NOS activity, protein NT residues and mRNA for the different NOS isoforms. In the presence of NADPH, WEMs from EAE-III rats showed lower Ca2+-dependent NOS activity than those from control group. These differences disappeared in the presence of exogenous calmodulin, flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH4) and NADPH. Of all the cofactors, just the omission of FAD caused comparable decrease of Ca2+-dependent NOS activity from both groups. Ca2+-independent NOS activity from EAE-III animals was insensitive to the omission of any of the cofactors, while in control animals this activity was significantly inhibited by the omission of either FAD or BH4. Increased levels of both iNOS mRNA and protein NT expression were observed in animals with EAE, which also showed lower levels of a thermolabile NOS inhibitor in WEM homogenates and sera than controls. In conclusion, during late EAE stages, constitutive Ca2+-dependent NOS activity decreases concomitantly with iNOS upregulation, which could be responsible for the high protein NT levels. The differential dependence of iNOS activity on cofactors and the absence of an endogenous thermolabile NOS inhibitor in animals with EAE could reflect additional control mechanisms of NOS activity in this model of multiple sclerosis.

Topics: Animals; Biopterins; Brain; Calcium; Calmodulin; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Flavin-Adenine Dinucleotide; Male; NADP; Neurons; Nitric Oxide; Nitric Oxide Synthase; Protein Isoforms; Rats; Rats, Inbred Lew; RNA, Messenger; Subcellular Fractions; Tyrosine

Nitric oxide (NO) derived from inducible NO synthase (iNOS) at sites of inflammation is closely related to host defense against infection and airway inflammation. Cytokines are known to stimulate NO production in human alveolar epithelial cells in a synergistic (nonlinear or nonadditive) manner. The mechanism of this synergy is not known. We measured the activation of the transcription factor NF-kappaB, the iNOS protein, and NO production in A549 monolayers (human alveolar epithelial cell line) in response to different combinations of IL-1beta, INF-gamma, and TNF-alpha (100 ng/ml), and the cofactors FMN, FAD, and BH4. We found that both IL-1beta and TNF-alpha could independently activate cytosolic NF-kappaB, direct its translocation into the nucleus, and induce iNOS monomer synthesis. In addition, different combinations of cytokines produced synergistic amounts of iNOS monomers. Exogenous BH4 (0.1 microM) had no impact on NO production induced by cytokine combinations that included IL-1beta, but significantly enhanced NO production in the presence of INF-gamma and TNF-alpha, and allowed TNF-alpha independently to produce NO. We conclude that there are at least three mechanisms of synergistic cytokine-induced NO production: (1) the biosynthesis of iNOS monomer due to nonlinear interactions by transcription factors, (2) synergistic cytosolic activation of NF-kappaB, and (3) parallel biosynthesis of BH4 in the presence of cytokine combinations that include IL-1beta.

Topics: Arginine; Biopterins; Cell Line, Transformed; Cell Nucleus; Cytokines; Epithelial Cells; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Humans; NF-kappa B; Nitric Oxide; Protein Transport; Pulmonary Alveoli

Effects of nicotine on arterial endothelium-dependent relaxations mediated by nitric oxide are controversial. Experiments were designed to test the hypothesis that nicotine can directly alter activity of endothelial nitric-oxide synthase (eNOS). NOS from aortic endothelial cells of untreated dogs and recombinant eNOS, neuronal NOS, and inducible NOS were used for these experiments. NOS activity was determined as conversion of L-[(3)H]arginine to L-[(3)H]citrulline in the absence or presence of nicotine (10(-7)-10(-3) M) in vitro. In separate assays, concentrations of cofactors NADPH, FAD, and tetrahydrobioprotein were reduced by half to assess for possible interaction with nicotine. With enzyme from aortic endothelial cells, total and calcium-dependent accumulation of citrulline increased by 30% in the presence of 10(-5) M nicotine. Nicotine dose dependently also increased citrulline accumulation by recombinant eNOS and neuronal NOS but not inducible NOS. Effects of nicotine on accumulation of citrulline by isolated eNOS and recombinant eNOS were further modulated by changes in the concentration of NADPH in the incubation solution. Our data demonstrate a significant effect of nicotine on eNOS-mediated citrulline accumulation. These results suggest that effects of nicotine on production of nitric oxide may depend on NADPH or oxygen radical interactions with NOS and thus may explain, in part, inconsistent findings of changes in production of endothelium-derived nitric oxide with nicotine administration.

Topics: Animals; Aorta, Thoracic; Arginine; Biopterins; Citrulline; Dogs; Endothelium, Vascular; Flavin-Adenine Dinucleotide; Male; NADP; Nicotine; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Recombinant Proteins

The present studies examined the hypothesis that the distribution of cerebral injury after a focal ischemic insult is associated with the regional distribution of nitric oxide synthase (NOS) activity.. Based on previous studies that certain anatomically well-defined areas are prone to become either core or penumbra after middle cerebral artery occlusion (MCAO), we measured NOS activity in these areas from the right and left hemispheres in a spontaneously hypertensive rat filament model. Four groups were studied: (1) controls (immediate decapitation); (2) 1.5 hours of MCAO with no reperfusion (R0); (3) 1.5 hours of MCAO with 0.5 hour of reperfusion (R0.5); and (4) 1.5 hours of MCAO with 24 hours of reperfusion (R24). Three groups of corresponding isoflurane sham controls were also included: 1.5 (S1.5) or 2 (S2.0) hours of anesthesia and 1.5 hours of anesthesia+24 hours of observation (S24).. Control core NOS activity for combined right and left hemispheres was 129% greater than penumbral NOS activity (P<0.05). Combined core NOS activity was also greater (P<0.05) in the three sham groups: 208%, 122%, and 161%, respectively. In the three MCAO groups, ischemic and nonischemic core NOS remained higher than penumbral regions (P<0.05). However, NOS activity was lower in the ischemic than in the nonischemic core in all three groups: R0 (29% lower), R0.5 (48%), and R24 (86%) (P<0.05). Addition of cofactors (10 micromol/L tetrahydrobiopterin, 3 micromol/L flavin adenine dinucleotide, and 3 micromol/L flavin mononucleotide) increased NOS activity in all groups and lessened the decrease in ischemic core and penumbral NOS.. Greater NOS activity in core regions could explain in part the increased vulnerability of that region to ischemia and could theoretically contribute to the progression of the infarct over time. The data also suggest that NOS activity during ischemia and reperfusion could be influenced by the availability of cofactors.

Topics: Anesthetics, Inhalation; Animals; Antioxidants; Arterial Occlusive Diseases; Biopterins; Brain Ischemia; Cerebral Arteries; Cricetinae; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Isoflurane; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Rats, Inbred SHR; Reperfusion; Reperfusion Injury; Time Factors

The fluorescence intensity of the two flavin prosthetic groups, FMN and FAD, in neuronal nitric oxide synthase (nNOS) was found to decay highly nonexponentially, being best described by four fluorescence lifetimes. This excited state heterogeneity is the result of multiple flavin quenching sites which are due to several flavin microenvironments created mainly by stacking with aromatic amino acids. Investigating nNOS in the absence of one or more of Ca2+/calmodulin, tetrahydrobiopterin, and heme revealed an influence of these cofactors on the microenvironments of the flavin prosthetic groups. Similar effects on the flavin rotational dynamics were found by analyzing the fluorescence anisotropy decay of the holo and of the different apo forms of nNOS. Since the tetrahydrobiopterin and the heme are located in the N-terminal oxygenase domain of nNOS, their effect on the flavins in the C-terminal reductase domain is explained by a folding back of the reductase domain onto the oxygenase domain. Thereby a domain-domain interface is created containing the FAD, FMN, heme, and tetrahydrobiopterin prosthetic groups which allows for efficient electron transfer during catalysis. The heme group, which is known to be essential for homodimerization of nNOS, was also found to be essential for the formation of the domain-domain interface.

Topics: Animals; Arginine; Biopterins; Brain; Calmodulin; Dimerization; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Fluorescence Polarization; Heme; Holoenzymes; Nerve Tissue Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Protein Binding; Protein Conformation; Rats; Spectrometry, Fluorescence; Substrate Specificity; Thermodynamics

To elucidate how thiols affect neuronal nitric oxide synthase (nNOS) we studied the binding of thiols to tetrahydrobiopterin (BH4)-free nNOS. Dithiothreitol (DTT), 2-mercaptoethanol, and L- and D-cysteine all bound to the heme with Kd values varying from 0.16 mM for DTT to 41 mM for L-cysteine. DTT, 2-mercaptoethanol, and L-cysteine yielded absorbance spectra with maxima at about 378 and 456 nm, indicative of bisthiolate complexes; the maximum at 426 nm with D-cysteine suggests binding of the neutral thiol. From the results with 2-mercaptoethanol we deduced that in 2-mercaptoethanol-free, BH4-free nNOS the sixth heme ligand is not a thiolate. DTT binding to nNOS containing one BH4 per dimer was biphasic. Apparently, the BH4-free subunit bound DTT with the same affinity as the BH4-free enzyme, whereas the BH4-containing subunit exhibited a > 100-fold lower affinity, indicative of competition between DTT and BH4 binding. Binding of DTT to the BH4-containing subunit was suppressed by L-arginine, whereas high-affinity binding was not affected, suggesting that L-arginine binds only to the BH4-containing subunit. DTT competitively inhibited L-citrulline production by nNOS containing one BH4 per dimer (Ki approximately 11 mM). Comparison of DTT binding and inhibition suggests that the heme of the BH4-free subunit is not involved in catalysis. Thermostability of nNOS was studied by preincubating the enzyme at various temperatures prior to activity determination. At nanomolar concentrations, nNOS was stable at 20 degrees C but rapidly deactivated at higher temperatures (t1/2 approximately 6 min at 37 degrees C). At micromolar concentrations, inactivation was 10 times slower. Absorbance and fluorescence measurements demonstrate that inactivation was not accompanied by major structural changes. The stabilization of nNOS by thiols was illustrated by the fact that omission of 2-mercaptoethanol during preincubation for 10 min at 30 degrees C led to an activity decrease of up to 90%.

Topics: Animals; Arginine; Biopterins; Calmodulin; Cysteine; Dithiothreitol; Enzyme Inhibitors; Enzyme Stability; Flavin-Adenine Dinucleotide; Heme; Mercaptoethanol; NADP; Neurons; Nitric Oxide Synthase; Protein Binding; Rats; Spectrometry, Fluorescence; Spectrophotometry; Sulfhydryl Compounds; Temperature

Using 14C-labeled arginine to 14C-labeled citrulline conversion assays in brain homogenates from 14- to 18-day-old and adult spontaneously hypertensive rats, we tested the hypotheses that maturation increases neuronal nitric oxide synthase (nNOS) activity and that this increase involves changes in cofactor availability and/or nNOS kinetics. nNOS activity (in pmol x mg(-1) x min(-1)) was 46% higher in adults (19 +/- 2) than in pups (13 +/- 1). The addition of 264 microM calmodulin (CaM), 3 microM FAD, 3 microM flavin adenine mononucleotide (FMN), and 10 microM tetrahydrobiopterin (BH4) increased NOS activity by 3, 46, 45, and 88% in pups and by 19, 40, 36, and 102% in adults, respectively. All cofactor effects were significant except for CaM in the pup homogenates. Cofactor effects were not significantly different between pup and adult homogenates, except for BH4, which increased absolute NOS activity more in adults than in pups. Values of maximal enzyme velocity (Vmax) for nNOS in the absence of added cofactors were greater in adults than in pups (104 +/- 5 vs. 53 +/- 3, P < 0.05). Addition of 3 microM FAD or 3 microM FMN increased pup Vmax values to 68 +/- 2 and 99 +/- 5, respectively, but had no effect in adults. BH4 did not affect Vmax in either group. Control values of the Michaelis-Menten constant (Km) for L-arginine were greater (P < 0.05) in pups (5.7 +/- 0.4 microM) than in adults (4.3 +/- 0.2 microM) and were significantly reduced by 10 microM BH4 to 3.8 +/- 0.2 and 2.9 +/- 0.1 microM, respectively. Neither FAD nor FMN affected Km values in either group. The results indicate that endogenous nNOS cofactor levels are not saturating in either pups or adults, changes in cofactor levels differentially affect NOS kinetics in pups and adults, and age-related differences in NOS activity result from fundamental differences in NOS kinetics. These findings support the general hypothesis that the increased vulnerability to ischemic stroke associated with maturation is due in part to corresponding increases in the capacity for nitric oxide synthesis.

Topics: Aging; Animals; Animals, Newborn; Biopterins; Brain; Calmodulin; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hypertension; Kinetics; Nitric Oxide Synthase; Rats; Rats, Inbred SHR

Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme-deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin.

Topics: Animals; Arginine; Binding Sites; Biopterins; Brain; Chromatography, Gel; Circular Dichroism; Citrulline; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Heme; Isoenzymes; Light; Macromolecular Substances; Neurons; Nitric Oxide Synthase; Nitroarginine; Protein Conformation; Protein Structure, Secondary; Rats; Scattering, Radiation; Thermodynamics

Topics: Animals; Baculoviridae; Biopterins; Brain; Cell Line; Chromatography, High Pressure Liquid; Coenzymes; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Heme; Nitric Oxide Synthase; Rats; Recombinant Proteins; Spectrophotometry; Spodoptera; Transfection

The increase in cyclic GMP contents in phototransduction is likely to be mediated with release of nitric oxide (NO). In this study we have characterized a constitutive NO synthase (NOS) isolated from rat retina supernatant. The activity of NOS was determined by monitoring L-citrulline formation from L-arginine. Soluble NOS from retina used L-arginine as substrate, with NADPH, tetrahydrobiopterin and FAD as cofactors. The enzyme activity was raised with additional calcium and was reduced with high concentrations of calmodulin inhibitors. Protein immunoblot and immunohistochemical analysis using a specific antibody against Type I NOS indicated that the same type of NOS is mainly localized in amacrine cells in the retina. However, the enzyme activity in freshly prepared sample was not completely abolished in the absence of calcium or with calmodulin inhibitors. In addition, NADPH diaphorase staining in retina was wider and more intensive than staining with the antibody against Type I NOS. These results indicate that rat retina contains more than one type of NOS.

Topics: Animals; Biopterins; Blotting, Western; Cross Reactions; Enzyme Inhibitors; Female; Flavin-Adenine Dinucleotide; Imidazoles; Immunohistochemistry; Male; NADP; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Rats; Rats, Wistar; Retina

Macrophage NO synthase (NOS) is a dimeric enzyme comprising two identical 130 kDa subunits and contains iron protoporphyrin IX (heme), tetrahydrobiopterin, FAD, FMN, and calmodulin. We have carried out limited proteolysis to locate the domains involved in prosthetic group binding and subunit interaction. Trypsin cleaved the subunits of dimeric macrophage NOS at a single locus, splitting the enzyme into two fragments whose denatured molecular masses were 56 and 74 kDa. The smaller fragments remained dimeric in their native form (112 kDa), contained heme and tetrahydrobiopterin, and could bind L-arginine, CO, or imidazole. In contrast, the larger fragments were monomeric in their native form, contained FAD, FMN, and CAM, and bound NADPH. Although neither purified fragment alone or in combination catalyzed NO synthesis from L-arginine, the flavin-containing fragment did catalyze cytochrome c reduction at a rate that was equivalent to that of native dimeric NOS. These results indicate that trypsin cuts macrophage NOS into two domains that can exist and function independently of one another. The domain that binds heme, H4biopterin, and substrate is also responsible for maintaining the NOS dimeric structure, while the domain containing FAD, FMN, and CAM is not required for subunit interaction. This suggests a structural model for macrophage NOS in which the subunits align in a head-to-head manner, with the oxygenase domains interacting to form a dimer and the reductase domains existing as independent extensions.

Topics: Amino Acid Oxidoreductases; Amino Acid Sequence; Animals; Biopterins; Calmodulin; Catalysis; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Heme; Macrophages; Mice; Molecular Sequence Data; Molecular Weight; Nitric Oxide Synthase; Peptide Fragments; Protein Conformation; Spectrum Analysis; Structure-Activity Relationship; Trypsin

Nitric oxide synthase (NOS) activity was measured, by the conversion of arginine to citrulline, in a preparation from the rat ileum consisting of the myenteric plexus and smooth muscle layers. A variety of incubating media were used in order to establish the optimal conditions required for the assay. NOS activity was present in the soluble fraction and was Ca(2+)- and calmodulin-dependent, characteristic of neuronal NOS. Exogenous Ca2+ was required for activity to be detectable but NOS activity progressively decreased with Ca2+ concentrations above 1.25 mM. Activity varied with arginine concentration, reaching saturation at 6 microM, and required the addition of the co-substrate NADPH. Endogenous levels of co-factors in the crude soluble fraction were not sufficient to maintain NOS activity. Omission of flavin adenine dinucleotide and tetrahydrobiopterin from the incubation medium reduced activity by 90%, and both co-factors had to be present for maximal activity to occur. These results emphasize the need to control assay conditions when measuring NOS activity in crude preparations from peripheral tissue.

Topics: Animals; Antioxidants; Arginine; Biopterins; Calcium; Calmodulin; Citrulline; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Ileum; In Vitro Techniques; Muscle, Smooth; Myenteric Plexus; NADP; Nitric Oxide Synthase; Rats; Rats, Wistar

Nitric oxide synthase (EC 1.14.23) was discovered in a Nocardia species. The bacterial nitric oxide synthase was purified as much as 380 fold by affinity chromatography over 2',5'-ADP-agarose. The partially purified enzyme required NADPH, O2, CA++, FAD, FMN, and tetrahydrobiopterin as cofactors in the conversion of L-arginine to L-citrulline and nitric oxide. The apparent Km for L-arginine was determined to be 8.2 microM, and the Vmax was 840 nmole NADPH consumed/min/mg protein. The enzyme was competetively inhibited by NG-nitro-arginine with an apparent Ki of 14.6 microM. The experimental evidence provides confirmation of the first microbial nitric oxide synthase in microorganisms.

Topics: Amino Acid Oxidoreductases; Arginine; Biopterins; Calcium; Citrulline; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; NADP; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nocardia; Oxygen

The cytokine-induced nitric oxide synthase (NOS) of macrophages is a homodimeric enzyme that contains iron protoporphorin IX (heme), FAD, FMN, tetrahydrobiopterin, and calmodulin. To investigate how the enzyme's quaternary structure relates to its catalytic activity and binding of prosthetic groups, dimeric NOS and its subunits were purified separately and their composition and catalytic properties compared. In contrast to dimeric NOS, purified subunits did not synthesize NO or contain bound heme or tetrahydrobiopterin. However, the subunits did contain FAD, FMN, and calmodulin in amounts comparable with dimeric NOS, displayed the light absorbance spectrum of an FAD- and FMN-containing flavoprotein, and generated an air-stable flavin semiquinone radical upon reduction of their ferricyanide-oxidized form. Dimeric NOS and NOS subunits were equivalent in catalyzing electron transfer from NADPH to cytochrome c, dichlorophenolindophenol, or ferricyanide at rates that were 8-30-fold faster than the maximal rate of NO synthesis by dimeric NOS. Reconstitution of subunit NO synthesis required their incubation with L-arginine, tetrahydrobiopterin, and stoichiometric amounts of heme and correlated with formation of a proportional amount of dimeric NOS in all cases. The dimeric NOS reconstituted from its subunits contained 0.9 heme and 0.44 tetrahydrobiopterin bound per subunit and had the spectral and catalytic properties of native dimeric NOS. Thus, NOS subunits are NADPH-dependent reductases that acquire the capacity to synthesize NO only through their dimerization and binding of heme and tetrahydrobiopterin. The ability of heme, tetrahydrobiopterin, and L-arginine to promote subunit dimerization is unprecedented and suggests novel roles for these molecules in forming and stabilizing the active dimeric NOS.

Topics: Amino Acid Oxidoreductases; Animals; Biopterins; Catalysis; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Heme; Macrophages; Mice; NADP; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Oxidoreductases; Substrate Specificity

Purified cerebellar nitric oxide (NO) synthase was found to reduce molecular oxygen to hydrogen peroxide at low concentrations of its substrate L-arginine or its cofactor tetrahydrobiopterin. The characteristics of oxygen reduction appeared to be similar to NO synthesis, as both reactions required reduced nicotinamide adenine dinucleotide phosphate (NADPH), were dependent on Ca2+/calmodulin, and showed optimal reaction rates at slightly acidic conditions. The electron transport from NADPH to molecular oxygen is probably mediated by the reduced flavins, flavine adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are bound in stoichiometrical amounts to the enzyme. NO synthase shows similarities to cytochrome P450 (cytochrome c) reductase, another FAD- and FMN-containing enzyme, and we found that NO synthase reduced cytochromes and artificial, low molecular mass electron acceptors in a superoxide dismutase-insensitive manner. Thus, NO synthase apparently represents a Ca(2+)-regulated, soluble isoform of cytochrome P450 reductase.

Topics: Amino Acid Oxidoreductases; Arginine; Biopterins; Cerebellum; Cytochrome c Group; Electron Transport; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hydrogen Peroxide; NADH Dehydrogenase; NADP; Nitric Oxide Synthase; Oxidation-Reduction; Oxygen

An endotoxin-induced form of nitric oxide synthase (EC 1.14.23) was purified to homogeneity from rat liver by sequential anion-exchange chromatography and affinity chromatography using 2',5'-ADP-Sepharose. The enzyme has a subunit molecular mass of 135 kDa as determined by SDS/PAGE, a maximum specific activity of 462 nmol of citrulline formed from arginine per min per mg, and a Km for arginine of 11 microM. The enzyme was strongly stimulated by the addition of calmodulin with an EC50 of 2 nM, but removal of free calcium from the assay medium only reduced activity by 15%. Calmodulin inhibitors significantly reduced the enzyme activity. Tetrahydrobiopterin, FAD, and FMN were all required for full enzyme activity. This form of endotoxin-induced nitric oxide synthase from liver differs from the inducible enzyme found in macrophages and is unusual in that it is stimulated by calmodulin with little dependence on the calcium ion concentration.

Topics: Amino Acid Oxidoreductases; Animals; Biopterins; Calmodulin; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Endotoxins; Enzyme Induction; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Kinetics; Klebsiella pneumoniae; Liver; Macromolecular Substances; Male; Molecular Weight; Nitric Oxide Synthase; Rats; Rats, Inbred Strains

Brain nitric oxide synthase is a Ca2+/calmodulin-regulated enzyme which converts L-arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H4biopterin). We found that purified NO synthase contains enzyme-bound H4biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H4biopterin was effective at substoichiometrical concentrations, these results indicate that NO synthase essentially depends on H4biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non-heme iron in equimolar amounts and exhibits striking activities, including a Ca2+/calmodulin-dependent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Biopterins; Calcium; Calmodulin; Cerebellum; Citrulline; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hydrogen Peroxide; Kinetics; NADP; Nitric Oxide; Nitric Oxide Synthase; Spectrophotometry; Swine