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Information on EC 1.14.13.39 - nitric-oxide synthase (NADPH) and Organism(s) Bos taurus and UniProt Accession P29473
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1.14.13.39 nitric-oxide synthase (NADPH)IUBMB Comments
The enzyme consists of linked oxygenase and reductase domains. The eukaryotic enzyme binds FAD, FMN, heme (iron protoporphyrin IX) and tetrahydrobiopterin, and its two domains are linked via a regulatory calmodulin-binding domain. Upon calcium-induced calmodulin binding, the reductase and oxygenase domains form a complex, allowing electrons to flow from NADPH via FAD and FMN to the active center. The reductase domain of the enzyme from the bacterium Sorangium cellulosum utilizes a [2Fe-2S] cluster to transfer the electrons from NADPH to the active center. cf. EC 1.14.14.47, nitric-oxide synthase (flavodoxin).
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- 1.14.13.39
- artery
- necrosis
- lipopolysaccharide
-
l-name
- lps
- tnf
- cardiovascular
- cox-2
- nitrite
- cyclooxygenase-2
- vessel
- hypertension
- cardiac
-
lps-induced
- dismutase
- cerebral
-
vasodilation
- nerve
- aortic
- ischemia
- pulmonary
- prostaglandin
- myocardial
- cgmp
- ng-nitro-l-arginine
- factor-alpha
- coronary
- acetylcholine
- wistar
-
endothelium-dependent
- tnf-alpha
- peroxynitrite
-
contractile
-
neuroprotective
-
lps-stimulated
-
reperfusion
- nf-kappab
-
nitrotyrosine
- microglia
- interferon-gamma
- nitroprusside
- ifn-gamma
-
vasoconstriction
-
guanylate
-
vasorelaxation
- il-1beta
-
lipopolysaccharide-induced
-
erectile
- endothelin-1
-
guanylyl
- pharmacology
- medicine
The taxonomic range for the selected organisms is: Bos taurus
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Synonyms
nos, inducible nitric oxide synthase, endothelial nitric oxide synthase, inducible no synthase, neuronal nitric oxide synthase, inducible nos, endothelial no synthase, endothelial nos, neuronal nos, no-synthase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
endothelium-derived relaxation factor-forming enzyme
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-
-
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endothelium-derived relaxing factor synthase
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-
-
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NADPH-diaphorase
-
-
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nitric oxide synthase
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-
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nitric oxide synthetase
-
-
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NO synthase
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-
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synthetase, nitric oxide
-
-
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-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 L-arginine + 3 NADPH + 3 H+ + 4 O2 = 2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
analysis of catalytic site by Fourier transform infrared spectroscopy, extent of modulation of vibrational modes upon photolysis of CO compound
2 L-arginine + 3 NADPH + 3 H+ + 4 O2 = 2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
isoform I and II are regulated by Ca2+/calmodulin, isoform II is Ca2+-independent, but requires calmodulin, inducible by cytokines
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
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oxidation
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-
-
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reduction
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PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
L-arginine,NADPH:oxygen oxidoreductase (nitric-oxide-forming)
The enzyme consists of linked oxygenase and reductase domains. The eukaryotic enzyme binds FAD, FMN, heme (iron protoporphyrin IX) and tetrahydrobiopterin, and its two domains are linked via a regulatory calmodulin-binding domain. Upon calcium-induced calmodulin binding, the reductase and oxygenase domains form a complex, allowing electrons to flow from NADPH via FAD and FMN to the active center. The reductase domain of the enzyme from the bacterium Sorangium cellulosum utilizes a [2Fe-2S] cluster to transfer the electrons from NADPH to the active center. cf. EC 1.14.14.47, nitric-oxide synthase (flavodoxin).
CAS REGISTRY NUMBER
COMMENTARY
125978-95-2
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
-
-
-
?
oxidized cytochrome c + NADPH + O2
reduced cytochrome c + NADP+ + H2O
-
-
-
?
L-homoarginine + NADPH + O2
?
-
constitutive endothelial membrane-bound and inducible soluble macrophage enzyme
-
-
?
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis
-
-
?
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
-
-
-
-
?
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
NO is an important signalling molecule, released by numerous cells, that acts in many tissues to regulate a diverse range of physiological and biological processes, including neurotransmission, immune defence and the regulation of apoptosis. NO plays a major role in the killing of intracellular pathogens as part of the innate immune response
-
-
?
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
-
the electron transfer between cofactors FMN and FAD is reversible
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
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-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
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-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
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physiological functions and pathophysiology of the isoforms
-
-
?
Ngamma-hydroxy-L-arginine + NADPH + O2
citrulline + NADP+ + NO
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best substrate
-
-
?
additional information
?
-
-
enzyme shows also superoxide formation activity, uneffected by L-arginine, inhibited by tetrahydrobiopterin and diphenyleneiodonium
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-
?
additional information
?
-
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enzyme shows also superoxide formation activity
-
-
?
additional information
?
-
-
endothelial NOS has a 6fold lower NO synthesis activity and 6-16fold lower cytochrome c reductase activity than neuronal NOS due to a significantly different electron transfer capacities, substrate specificity and mechanism, oveview
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis
-
-
?
additional information
?
-
-
enzyme shows also superoxide formation activity
-
-
?
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
-
-
-
-
?
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
NO is an important signalling molecule, released by numerous cells, that acts in many tissues to regulate a diverse range of physiological and biological processes, including neurotransmission, immune defence and the regulation of apoptosis. NO plays a major role in the killing of intracellular pathogens as part of the innate immune response
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
-
-
-
?
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
-
physiological functions and pathophysiology of the isoforms
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Calmodulin
in the absence of calmodulin, the wild type enzyme activity is less than 15% of the maximum calmodulin-dependent values
Calmodulin
maximum calmodulin-dependent activity is measured at 1.5 mM CaCl2, phosphorylation within an autoinhibitory domain in endothelial nitric oxide synthase reduces the Ca2+ concentrations required for calmodulin to bind and activate the enzyme
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
required for calmodulin to bind and activate the enzyme, maximum calmodulin-dependent activity is measured at 1.5 mM CaCl2
Ca2+
Ca2+
-
constitutive endothelial and inducible membrane bound macrophage enzyme are strictly Ca2+-dependent
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1-phenylimidazole
-
reversible inhibition of endothelial enzyme, competitive versus L-arginine and tetrahydrobiopterin, no inhibition of cytochrome c reduction
7-nitroindazole
-
reversible inhibition of endothelial enzyme, competitive versus tetrahydrobiopterin, no inhibition of cytochrome c reduction
diphenylene iodonium
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inhibition of superoxide production of recombinant isoform III
ethylene glycol bis(beta-amino-ethylether)-N,N,N',N'-tetraacetic acid
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i.e. EGTA, complete inhibition of cytosolic enzyme, partial inhibition of particulate enzyme
imidazole
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inhibition of the endothelial enzyme, competitive versus L-arginine, no inhibition of cytochrome c reduction
additional information
-
Ngamma,Ngamma'-dimethyl-L-arginine has no inhibitory effect
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Ngamma-monomethyl-L-arginine
-
inhibits citrulline formation, not cytochrome c reduction
Ngamma-nitro-L-arginine
-
inhibits citrulline formation, not cytochrome c reduction
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
heat shock protein 90
heat shock protein 90 enhances the affinity of wild-type enzyme to NADPH, L-arginine, and calmodulin but not to Ca2+ and BH4
-
additional information
iNOS is induced by pathogens and their components, e.g. induction in alveolar macropages by infection with the intracellular pathogen Mycobacterium bovis
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additional information
-
iNOS is induced by pathogens and their components, e.g. induction in alveolar macropages by infection with the intracellular pathogen Mycobacterium bovis
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
kinetics of cytcochrome c reduction and of reaction with FMN and cytochrome c, overview, eNOS and nNOS differ slightly in their NADP(H) interaction and flavin thermodynamics, and show distinct electron transfer activities, two-state conformational equilibrium of the FMN subdomain, overview
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
low eNOS activity might involve an altered interaction with NADP(H)
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.4
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
-
3 isoforms: 1. neuronal, soluble isoform I is constitutively expressed in brain and other tissues and Ca2+-regulated, 2. soluble isoform II is usually not constitutively expressed, but inducible in macrophages and other cells, 3. isoform III is membrane-bound and expressed in endothelial cells
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
constitutive endothelial enzyme: predominantly membrane-bound, inducible macrophage enzyme: equally distributed between cytosol and membrane, small constitutive membrane-bound portion in murine macrophages
additional information
-
3 isoforms: 1. neuronal, soluble isoform I is constitutively expressed in brain and other tissues and Ca2+-regulated, 2. soluble isoform II is usually not constitutively expressed, but inducible in macrophages and other cells, 3. isoform III is membrane-bound and expressed in endothelial cells
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). NOS3_BOVIN
1205
0
133287
Swiss-Prot
other Location (Reliability: 2)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
150000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
additional information
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
S1179D
S617D
the phosphomimetic substitution doubles the maximum calmodulin-dependent enzyme activity and decreases the EC50(Ca2+) values for calmodulin binding and enzyme activation compared to the wild type enzyme
S617D/S1179D
the mutation doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC50(Ca2+) values for calmodulin binding and enzyme activation
S617D/S635D
the maximum calmodulin-dependent synthase activity of S617/635D eNOS is about 2fold higher than the wild type activity
additional information
S1179D
the phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC50(Ca2+) values for calmodulin binding and enzyme activation about 35%
S1179D
mutation augments enzyme activity and increases its sensitivity to heat shock protein 90 (Hsp90). Mutation of Ser1179 to aspartic acid plays a dominant role in changing the sensitivity of the enzyme to NADPH.
additional information
deletion of the autoinhibitory domain doubles the maximum calmodulin-dependent enzyme activity and decreases the EC50(Ca2+) values for calmodulin binding and calmodulin-dependent enzyme activation
additional information
-
deletion of the autoinhibitory domain doubles the maximum calmodulin-dependent enzyme activity and decreases the EC50(Ca2+) values for calmodulin binding and calmodulin-dependent enzyme activation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-85°C, 50 mM Tris (pH 7.4) with 10% (v/v) glycerol, 100 mM NaCl, 0.2 mM L-arginine, 0.0002 mM 5,6,7,8-tetrahydro-L-biopterin, 0.02 mM dithiothreitol, and 1 mM 2',3'-AMP (mixed isomers), up to 1 month, no detectable loss of catalytic activity
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-NTA Sepharose column chromatography and 2'-5' ADP Sepharose resin column chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
overexpression of wild-type endothelial enzyme and mutant endothelial enzyme S1179D in Escherichia coli
expression of endothelial isoform III in Spodoptera frugiperda cells via baculovirus infection
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iNOS, DNA and amino acid sequence determination and analysis, phylogenetic analysis
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ohshima, H.; Oguchi, S.; Adachi, H.; Iida, S.; Suzuki, H.; Sugimura, T.; Esumi, H.
Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution
Biochem. Biophys. Res. Commun.
183
238-244
1992
Bos taurus, Rattus norvegicus, Rattus norvegicus Sprague-Dawley
Hecker, M.; Walsh, D.T.; Vane, J.R.
On the substrate specificity of nitric oxide synthase
FEBS Lett.
294
221-224
1991
Bos taurus, Mus musculus
Wolff, D.J.; Datto, G.A.
Identification and characterization of a calmodulin-dependent nitric oxide synthase from GH3 pituitary cells
Biochem. J.
285
201-206
1992
Bos taurus
Foerstermann, U.; Closs, E.I.; Pollock, J.S.; Nakane, M.; Schwarz, P.; Gath, I.; Kleinert, H.
Nitric oxide synthase isoenzymes. Characterization, purification, molecular cloning, and functions
Hypertension
23
1121-1131
1994
Bos taurus, Homo sapiens, Mus musculus, Rattus norvegicus, Sus scrofa
Nam, S.W.; Seo, D.W.; Sung, D.S.; Han, J.W.; Hong, S.Y.; Lee, H.W.
Nitric oxide synthase from bovine pancreas: purification and characterization
Arch. Pharm. Res.
21
128-134
1998
Bos taurus
Wever, R.M.F.; Van Dam, T.; Van Rijn, H.J.M.; De Groot, F.; Rabelink, T.J.
Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase
Biochem. Biophys. Res. Commun.
237
340-344
1997
Bos taurus
Wolff, D.J.; Lubeskie, A.; Umansky, S.
The inhibition of the constitutive bovine endothelial nitric oxide synthase by imidazole and indazole agents
Arch. Biochem. Biophys.
314
360-366
1994
Bos taurus
Presta, A.; Liu, J.; Sessa, W.C.; Stuehr, D.J.
Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymic activity
Nitric Oxide
1
74-87
1997
Bos taurus
Ingledew, W.J.; Smith, S.M.; Gao, Y.T.; Jones, R.J.; Salerno, J.C.; Rich, P.R.
Ligand, cofactor, and residue vibrations in the catalytic site of endothelial nitric oxide synthase
Biochemistry
44
4238-4246
2005
Bos taurus (P29473), Bos taurus
Ilagan, R.P.; Tiso, M.; Konas, D.W.; Hemann, C.; Durra, D.; Hille, R.; Stuehr, D.J.
Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins
J. Biol. Chem.
283
19603-19615
2008
Bos taurus
Widdison, S.; Ashley, G.R.; Howard, C.J.; Coffey, T.J.
Characterisation of bovine inducible nitric oxide synthase
Vet. Immunol. Immunopathol.
117
302-309
2007
Bos taurus (Q9BDQ7), Bos taurus
Tran, Q.K.; Leonard, J.; Black, D.J.; Persechini, A.
Phosphorylation within an autoinhibitory domain in endothelial nitric oxide synthase reduces the Ca2+ concentrations required for calmodulin to bind and activate the enzyme
Biochemistry
47
7557-7566
2008
Bos taurus (P29473), Bos taurus
Tran, Q.K.; Leonard, J.; Black, D.J.; Nadeau, O.W.; Boulatnikov, I.G.; Persechini, A.
Effects of combined phosphorylation at Ser-617 and Ser-1179 in endothelial nitric-oxide synthase on EC50(Ca2+) values for calmodulin binding and enzyme activation
J. Biol. Chem.
284
11892-11899
2009
Bos taurus (P29473)
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