Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.19 (NOS)
 7,285 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mouse macrophage NO-synthase (mNOS) was expressed in a unique yeast-based system by using a three-step procedure which allows yeast growth and NOS expression to be uncoupled. Despite cytotoxic effects related to mNOS expression, levels of catalytically active enzyme up to 0.5 mg of protein per 5 L of culture was obtained after purification. Its electrophoretic, spectroscopic [lambda max = 446 nm for its Fe(II)-CO complex], and catalytic properties were similar to those previously reported for mNOS purified from macrophages. Recombinant mNOS catalyzed the NADPH-dependent oxidation of L-arginine to citrulline (Km = 7 +/- 3 microM) as well as the reduction of cytochrome C by NADPH [Km = 34 +/- 8 microM and Vm = 25 +/- 5 mumol min-1 (mg of protein-1)]. Two mutants of mNOS in which Cys 194 was replaced with either serine or histidine were constructed and expressed in the same yeast strain at a level higher than that of the wild type protein, as they appear less toxic for the host. Both mutants exhibited electrophoretic properties and activities toward cytochrome C reduction identical to those of wild type NOS. However, they were unable to catalyze the oxidation of L-arginine to citrulline and did not appear to bind heme (no appearance of peaks around 400 and 446 nm for the resting enzyme and its CO complex, respectively, in visible spectroscopy). These data provide the first experimental evidence in favor of previous suggestions that Cys 194 was the proximal iron ligand of mouse mNOS.
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PMID:Expression in yeast and purification of functional macrophage nitric oxide synthase. Evidence for cysteine-194 as iron proximal ligand. 867 49

An investigation of the changes in size, number and distribution of NOS-containing neuronal somata in the rat kidney was undertaken. The immunoperoxidase method for the staining of NOS and the histochemical method for the demonstration of NADPH-d were applied to serial thick sections (100 microns) of whole kidneys. Animals at embryonic day 14 (ED14), ED16, ED18, ED20, at birth (PD0), and at postnatal days 4 (PD4), PD12, PD21 and PD35 were studied. NOS-containing neuronal somata were observed by the 20th day of gestation in some kidneys and were consistently seen at birth. They were usually seen in groups of separated neuronal somata or in tight clusters. The neuronal somata were often attached or embedded in nerve bundles. As the kidney developed, the number of neuronal somata separated from each other increased, while the number of clusters remained relatively constant. The size of the neuronal somata increased with development. There were highly significant statistical differences in the size of the neuronal somata between all groups, except between PD12 and PD21. The distribution of neuronal somata at birth was similar to that of the adult. They could be found, (a) at the free renal pelvic wall; (b) in the connective tissue at the angular space between the renal pelvis and the renal parenchyma (SPP); and (c) along the interlobar vessels. At birth and in the early stages of development, the greatest number of neuronal somata were located at the renal pelvis. In the later stages of development, more neuronal somata appear in the connective tissue between the renal pelvis and the renal parenchyma. The location of NOS-containing neuronal somata suggests that they might have a modulatory role on the sympathetic and sensory renal nerves all through development.
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PMID:Development of NOS-containing neuronal somata in the rat kidney. 874 Jun 64

1. The expression of neuronal isoform of nitric oxide synthase (nNOS) was studied in human retinal tissues. The cDNA sequence was cloned in human retinal poly (A)+ RNA by the RT-PCR method and encompassed an open-reading frame of 4,302 bp encoding 1,434 amino acids. This sequence showed a possibility of genetic polymorphism in comparison to human brain form. 2. Restriction fragment length polymorphism (RFLP) patterns of a partial cDNA fragment suggest that there is genetic polymorphism in the neuronal form of NOS. Important differences were observed in a certain region between human retinal and brain froms. This region is a result of frame shift by the addition of three cytidines. In this study, regions from human brain (cerebellum) and skeletal muscle as well as retina were sequenced to confirm the difference in this region. The sequences from these tissues were completely identical. This indicated that genetic polymorphism of nNOS gene was due to single base substitution and not frame shift phenomenon by addition or deletion of bases. 3. The nNOS mRNA of approximately 12 kb was detected by northern blot analysis. The lower level of the expression was distinguished in comparison to those of human brain and skeletal muscle. The cDNA transiently transfected into CHO-K1 cells expressed a protein which contained a significant level of NOS activity. The size of the nNOS was found to be approximately 160 kDa by both in vitro and in vivo translation systems. This NOS was calcium dependent and the K(m) for arginine was 4.4 microM. 4. The Ca+2, L-arginine and NADPH dependency along with the inhibitory effect of N-nitro-L-arginine on NOS activity were evaluated. The finding of a constitutive from of NOS in human retina, which is calcium-NADPH dependent, gives further credence to the possible role of nitric oxide in retinal function and neuronal diseases.
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PMID:Neuronal isoform of nitric oxide synthase is expressed at low levels in human retina. 887 52

The effect of a neuroleptic, haloperidol (HP), on nitric oxide formation catalyzed by neuronal nitric oxide synthase (n-NOS) in the porcine brain was investigated. HP inhibited n-NOS activity noncompetitively versus L-arginine as a substrate, decreasing the maximal velocity (Ki value for HP = 31 microM). HP also inhibited the CaM-dependent NADPH consumption by n-NOS (IC50 = 221 microM). These data demonstrate the possibility that HP may mediate some neuronal functions through inhibiting NO release by preventing either the electron transfer through n-NOS or the formation of the activated reduced species of oxygen necessary for the formation of citrulline. And an interaction of HP with CaM may possibly affect the consumption of NADPH and n-NOS enzyme activity.
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PMID:Haloperidol inhibits neuronal nitric oxide synthase activity by preventing electron transfer. 892 32

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.
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PMID:Human endothelial nitric oxide synthase: expression in Escherichia coli, coexpression with calmodulin, and characterization. 895 Oct 46

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (.NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify .NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable .NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal .NO standards were only partially recovered and the dismutation of superoxide (O2-.), which otherwise scavenges. .NO to yield ONOO-, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO- resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8-1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to .NO or that .NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal .NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO-) to .NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from .NO decomposition, was consistent with formation of an .NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that .NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.
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PMID:No .NO from NO synthase. 896 79

Endothelial nitric-oxide synthase (eNOS) is comprised of two identical subunits. Each subunit has a bidomain structure consisting of an N-terminal oxygenase domain containing heme and tetrahydrobiopterin (BH4) and a C-terminal reductase domain containing binding sites for FAD, FMN, and NADPH. Each subunit is also myristoylated and contains a calmodulin (CaM)-binding site located between the oxygenase and reductase domains. In this study, wild-type and mutant forms of eNOS have been expressed in a baculovirus system, and the quaternary structure of the purified enzymes has been analyzed by low temperature SDS-PAGE. eNOS dimer formation requires incorporation of the heme prosthetic group but does not require myristoylation or CaM or BH4 binding. In order to identify domains of eNOS involved in subunit interactions, we have also expressed eNOS oxygenase and reductase domain fusion proteins in a yeast two-hybrid system. Corresponding human neuronal NOS (nNOS) and murine inducible NOS (iNOS) fusion proteins have also been expressed. Comparative analysis of NOS domain interactions shows that subunit association of eNOS and nNOS involves not only head to head interactions of oxygenase domains but also tail to tail interactions of reductase domains and head to tail interactions between oxygenase and reductase domains. In contrast, iNOS subunit association involves only oxygenase domain interactions.
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PMID:Subunit interactions of endothelial nitric-oxide synthase. Comparisons to the neuronal and inducible nitric-oxide synthase isoforms. 899 32

N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitric- oxide synthase (iNOS). The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s-1 and a binding constant of 2.0 microM. Inhibition was dependent on the cofactor NADPH. L-Arginine was a competitive inhibitor of 1400W binding with a Ks value of 3.0 microM. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a Kd value </= 7 nM. In contrast, inhibition of human neuronal NOS and endothelial NOS (eNOS) was relatively weaker, rapidly reversible, and competitive with L-arginine, with Ki values of 2 microM and 50 microM, respectively. Thus, 1400W was at least 5000-fold selective for iNOS versus eNOS. This selectivity was similar to that observed in rat aortic rings, in which 1400W was greater than 1000-fold more potent against rat iNOS than eNOS. Finally, 1400W was greater than 50-fold more potent against iNOS than eNOS in a rat model of endotoxin-induced vascular injury. Thus, the potency and selectivity of 1400W inhibition of iNOS both in vitro and in vivo were far greater than of any previously described iNOS inhibitor.
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PMID:1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. 903 May 56

The cell population and distribution of NADPH-diaphorase positive and NOS immunoreactive intramural ganglion cells were examined on stretched whole-mount preparations of the guinea pig urinary bladder which was divided into 3 regions: base, body and dome. The results showed that the highest frequency both of NADPH-d and NOS positive neurons was observed in the bladder base. Cell counts in the whole bladder showed that the number of NADPH-d positive neurons was much more than that of NOS immunoreactive neurons. Using neuron specific enolase (NSE) positive neurons as a reference (100%), NADPH-d positive neurons accounted for 84% while NOS immunoreactive neurons only made up 45% of the total neuronal population. These results, along with previous studies on the function of nitric oxide, suggest that nitric oxide may be involved in the relaxation activity in the bladder base during micturition. The significant difference in the number of NADPH-d positive and NOS immunoreactive neurons suggests that the localisation of one enzyme does not necessarily reflect the presence of the other.
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PMID:Distribution of NADPH-diaphorase and nitric oxide synthase-containing neurons in the intramural ganglia of guinea pig urinary bladder. 903 89

Nitric oxide synthase (EC 1.14.13.39) is a homodimer. Limited proteolysis has previously shown that it consists of two major domains. The C-terminal or reductase domain binds FMN, FAD and NADPH. The N-terminal or oxygenase domain is known to bind arginine, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and haem. The exact residues of the inducible nitric oxide synthase (iNOS) protein involved in binding to these molecules have yet to be identified, although the haem moiety is known to be co-ordinated through a cysteine thiolate ligand. We have expressed two forms of the haem-binding domain of human iNOS (residues 1-504 and 59-504) in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The iNOS 1-504 and 59-504 fusion proteins bound similar amounts of haem, Nomega-nitro-l-arginine (nitroarginine) and tetrahydrobiopterin, showing that the first 58 residues are not required for binding these factors. Using site-directed mutagenesis we have mutated Cys-200, Cys-217, Cys-228, Cys-290, Cys-384 and Cys-457 to alanine residues within the iNOS 59-504 haem-binding domain. Mutation of Cys-200 resulted in a complete loss of haem, nitroarginine and tetrahydrobiopterin binding. Mutants of Cys-217, Cys-228, Cys-290, Cys-384 or Cys-457 showed no effect on the haem content of the fusion protein, no effect on the reduced CO spectral peak (444 nm) and were able to bind nitroarginine and tetrahydrobiopterin at levels equivalent to the wild-type fusion protein. After removal of the GST polypeptide, the wild-type iNOS 59-504 domain was dimeric, whereas the C200A mutant form was monomeric. When the mutated domains were incorporated into a reconstructed full-length iNOS protein expressed in Xenopus oocytes, only the Cys-200 mutant showed a loss of catalytic activity: all the other mutant iNOS proteins showed near wild-type enzymic activity. From this systematic approach we conclude that although Cys-217, Cys-228, Cys-290, Cys-384 and Cys-457 are conserved in all three NOS isoforms they are not essential for cofactor or substrate binding or for enzymic activity of iNOS, and that Cys-200 provides the proximal thiolate ligand for haem binding in human iNOS.
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PMID:Cysteine-200 of human inducible nitric oxide synthase is essential for dimerization of haem domains and for binding of haem, nitroarginine and tetrahydrobiopterin. 917 73


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