Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:1.5.1.19 (NOS)
7,285 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A gene coding for rat neuronal nitric oxide synthase (nNOS) has been cloned into pCWori and the vector has been expressed in Escherichia coli. The expressed enzyme has been purified with a final yield of purified protein of approximately 1 mg/g of wet cells. The recombinant protein reconstituted with calmodulin and Ca2+ exhibits spectroscopic and catalytic properties identical to those reported in the literature for nNOS. Reaction of recombinant nNOS with phenyldiazene produces a phenyl-iron (Fe.Ph) complex with a maximum at 470 nm. Formation of this complex is paralleled by inactivation of the enzyme and is inhibited by arginine, the natural substrate of the enzyme. Phenyl-iron complex formation does not alter the rate of electron transfer from the flavin domain to cytochrome c. Addition of ferricyanide triggers migration of the phenyl residue from the iron to the porphyrin nitrogens. The N-phenylprotoporphyrin isomers with the phenyl on the nitrogens of pyrrole rings B, A, C, and D are formed in, respectively, approximately a 14:20:21:45 ratio. The regioisomer pattern indicates that the active site of NOS is open to some extent above all four pyrrole rings but more so above pyrrole ring D. Arylhydrazines are thus not only a new class of inhibitors of nNOS but provide useful information on the active site topology of the enzyme.
...
PMID:Neuronal nitric oxide synthase. Expression in Escherichia coli, irreversible inhibition by phenyldiazene, and active site topology. 754 92

A baculovirus system was used to express the oxygenase and reductase domains of human endothelial nitric-oxide synthase (ecNOS) as distinct proteins. The oxygenase domain (residues 1-491) was expressed using a vector containing a His6 tag at the N terminus. The purified oxygenase domain had an apparent molecular mass of approximately 54 kDa, and retained the ability to bind L-arginine and form the ferrous CO complex. The purified reductase domain (residues 492-1244) had an apparent molecular mass of approximately 82 kDa and retained the ability to catalyze NADPH-dependent cytochrome c reduction, which was enhanced 10-fold by the presence of Ca2+/calmodulin. Both purified domains exhibited immunoreactivity to rabbit anti-ecNOS IgG. The NOS activity was successfully reconstituted by mixing the two domains. These results demonstrate for the first time that the two domains of ecNOS are catalytically intact and can be reconstituted in vitro.
...
PMID:Endothelial nitric-oxide synthase. Evidence for bidomain structure and successful reconstitution of catalytic activity from two separate domains generated by a baculovirus expression system. 866 33

Human endothelial nitric oxide synthase (eNOS) has been cloned and expressed in Escherichia coli. The spectroscopic properties and specific activity (100-130 nmol x min(-1) x mg(-1) at 37 degrees C) of the recombinant protein are similar to those of the bovine enzyme. FPLC and low-temperature SDS-PAGE indicate that the protein is mostly dimeric in both the absence and presence of tetrahydrobiopterin. Human eNOS thus has a higher tendency to dimerize than the bovine enzyme. A chloramphenicol-resistant, trc promoter-based plasmid has been constructed that allows coexpression of human calmodulin (CaM). Coexpression of CaM increases more than threefold the amount of expressed eNOS, stabilizes the recombinant protein, and significantly augments its specific activity (to 140-170 nmol x min(-1) x mg(-1) at 37 degrees C). The cytochrome c reduction activity is also improved by CaM coexpression. These increases in activity are not achieved by the addition of CaM to eNOS expressed in the absence of CaM. Gel filtration studies suggest that CaM coexpression produces a more elongated eNOS structure and alters the NADPH binding domain. CaM coexpression has been shown previously to be required for successful expression of the inducible NOS isoform, but this is the first demonstration that CaM coexpression improves the expression of a constitutive isoform.
...
PMID:Human endothelial nitric oxide synthase: expression in Escherichia coli, coexpression with calmodulin, and characterization. 895 Oct 46

The anti-estrogen drug tamoxifen (TMX) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (nNOS) (IC50 = 2 +/- 0.5 microM), whereas it was inactive toward inducible macrophage NOS (IC50 > 100 microM). TMX affected the activation of NOS by calmodulin, as it not only inhibited L-arginine oxidation to nitric oxide and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by nNOS. These results suggest that TMX could exert some of its biological effects by interfering with constitutive NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.
...
PMID:Strong inhibition of neuronal nitric oxide synthase by the calmodulin antagonist and anti-estrogen drug tamoxifen. 946 53

It has been proposed that Cys99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH4) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys331 to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation with L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored. In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm. C331A-nNOS, as isolated, did not bind detectable levels of either [3H]Nomega-nitro-L-arginine or [3H]BH4, but [3H]BH4 binding was reinstated after extended incubation with excess L-arginine. On the other hand, C331A-nNOS and WT-NOS were identical with regard to imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2, 6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM). The estimated Kd for L-arginine binding to C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 microM versus 2-3 microM). Here we propose that Cys331 plays an important role in stabilizing L-arginine binding to nNOS. Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH4-substrate binding interactions as broadcast from this mutated cysteine residue. Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for normalized BH4 binding and nitric oxide synthetic activity.
...
PMID:The C331A mutant of neuronal nitric-oxide synthase is defective in arginine binding. 985 5

The heme of neuronal nitric-oxide synthase participates in oxygen activation but also binds self-generated NO during catalysis resulting in reversible feedback inhibition. We utilized point mutagenesis to investigate if a conserved tryptophan residue (Trp-409), which engages in pi-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Surprisingly, mutants W409F and W409Y were hyperactive compared with the wild type regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg, NADPH oxidation measurements showed that electron flux through the heme was actually slower in the Trp-409 mutants than in wild-type nNOS. However, little or no NO complex accumulated during NO synthesis by the mutants, as opposed to the wild type. This difference was potentially related to mutants forming unstable 6-coordinate ferrous-NO complexes under anaerobic conditions even in the presence of Arg and tetrahydrobiopterin. Thus, Trp-409 mutations minimize NO feedback inhibition by preventing buildup of an inactive ferrous-NO complex during the steady state. This overcomes the negative effect of the mutation on electron flux and results in hyperactivity. Conservation of Trp-409 among different NOS suggests that the ability of this residue to regulate heme reduction and NO complex formation is important for enzyme physiologic function.
...
PMID:Tryptophan 409 controls the activity of neuronal nitric-oxide synthase by regulating nitric oxide feedback inhibition. 1048 Sep

A water-soluble iron complex with N-dithiocarboxysarcosine (Fe-DTCS) has been developed as an ESR spin-trapping agent for NO and successfully applied to ESR imaging of endogenous NO production in mice. We attempted to measure NO produced by purified neuronal NO synthase (nNOS) by this method, but could not detect NO. We speculated that Fe-DTCS inhibits NOS activity. In fact, it markedly inhibited NOS activity with an IC50 value of 9.7 +/- 0.7 microM in the citrulline-formation assay. DTCS alone did not inhibit the activity. An iron complex with N-methyl-D-glucamine dithiocarbamate, a similar spin-trapping agent for NO, also inhibited the activity, with an IC50 value of 25.1 +/- 2.9 microM. Fe-DTCS suppressed cytochrome c and ferricyanide reductase activities of nNOS, and markedly increased nNOS-mediated NADPH oxidation. Concomitantly, it accelerated oxygen consumption caused by activated nNOS. These results suggest that the ESR spin-trapping agent Fe-DTCS inhibits NO synthesis by interfering with the physiological electron flow from NADPH to nNOS heme iron.
...
PMID:Reaction of neuronal nitric oxide synthase with the nitric oxide spin-trapping agent, iron complexed with N-dithiocarboxysarcosine. 1058 70

Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K(m)/EC(50) for L-arginine, calmodulin, and calcium, whereas there was a 2-fold increase in the rate of NO production for S1179D and a 2-4-fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.
...
PMID:Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation. 1069 2

It has been postulated that a segment (residues 594-645) inserted in the FMN subdomain of human endothelial nitric-oxide synthase (eNOS) plays a crucial role in controlling Ca(2+)-dependent CaM binding for eNOS activity. To investigate its functions, we expressed human eNOS in a baculovirus system with deletion of a 45-residue segment from this region (residues 594-606 and 614-645, designated as Delta45eNOS), and characterized the purified mutant enzyme. In contrast with wild-type eNOS, Delta45eNOS exhibited characteristics resembling inducible NOS (iNOS). It contained an endogenously bound CaM, which was essential in folding and stabilizing this mutant enzyme, and retained 60% of L-citrulline formation in 5 mM EGTA. We also produced four N-terminally truncated reductase domains with or without the 45-residue segment, and either including or excluding the CaM-binding sequence. Basal cytochrome c reductase activity of reductase domains without the 45-residue segment was up to 20 fold greater than that of corresponding insert-containing domains, and higher than CaM-stimulated activity of the wild-type enzyme. A series of mutants with smaller fragment deletion in this region such as Delta594-604, Delta605-612, Delta613-625, Delta626-634, Delta632-639, and Delta640-645 mutants were further characterized. The crude lysate of mutants Delta613-625 and Delta632-639 did not show activity in the presence of Ca(2+)/CaM, while other four mutants had activity comparable to that of WTeNOS. The purified Delta594-604 and Delta605-612 proteins had a 3-5-fold higher affinity for Ca(2+)/CaM, but their L-citrulline forming activity was still 80% dependent upon the addition of Ca(2+)/CaM. Both mutants exhibited a low level of the cytochrome c and ferricyanide reductase activities, which either did not respond to (Delta594-604) or slightly enhanced by (Delta605-612) the exogenous CaM. In contrast, activities of Delta626-634 and Delta640-645 like those of WTeNOS were largely Ca(2+)/CaM-dependent. Thus, our findings indicate that the N-terminal half of the 594-645 segment containing residues 594-612 plays a significant role in regulating Ca(2+)/CaM binding.
...
PMID:Characterization of the roles of the 594-645 region in human endothelial nitric-oxide synthase in regulating calmodulin binding and electron transfer. 1077 22

Iodonium compounds, especially diphenylene iodonium and iodonium diphenyl are used extensively as inhibitors of NADH-ubiquinone reductase and NADPH oxidase activity. Here, the use of a new iodonium compound, phenoxaiodonium is reported. The IC(50) of neutrophil superoxide production, measured using the superoxide dismutase inhibitable rate of cytochrome c reduction, was approximately 0.75 microM, while 50% inhibition of mitochondrial respiration, measured by the rate of oxygen uptake using a Clark type oxygen electrode, was at approximately 20 microM. The inhibition of oxidation of xanthine to urate by xanthine oxidase was also studied, giving a K(i) of 0.2 microM. Inhibition of nitric oxidase synthase (NOS: from rat brain) by 0.2 microM phenoxaiodonium was equivalent to 1 mM N(G)-nitro-L-arginine methyl ester HCl (L-NAME), that is total abolition of activity. We conclude that phenoxaiodonium is an extremely good inhibitor of flavo-enzymes, but like diphenylene iodonium and iodonium diphenyl, will be of limited use as a pharmacological tool for the elucidation of the involvement of such enzymes in specific cellular functions.
...
PMID:The inhibition of flavoproteins by phenoxaiodonium, a new iodonium analogue. 1092 15


1 2 3 4 Next >>

---