Characterization Of Nitric Oxide Synthase Research Articles

Purification and Characterization of Nitric Oxide Synthase from Bovine Kidney and Investigating Drug-Induced Toxicities of Some Antibiotics on the Enzyme Activity

Purification and Characterization of Nitric Oxide Synthase from Bovine Kidney and Investigating Drug-Induced Toxicities of Some Antibiotics on the Enzyme Activity

Identification and molecular characterization of nitric oxide synthase (NOS) gene in the intertidal copepod Tigriopus japonicus

In copepods, no information has been reported on the structure or molecular characterization of the nitric oxide synthase (NOS) gene. In the intertidal copepod Tigriopus japonicus, we identified a NOS gene that is involved in immune responses of vertebrates and invertebrates. In silico analyses revealed that nitric oxide (NO) synthase domains, such as the oxygenase and reductase domains, are highly conserved in the T. japonicus NOS gene. The T. japonicus NOS gene was highly transcribed in the nauplii stages, implying that it plays a role in protecting the host during the early developmental stages. To examine the involvement of the T. japonicus NOS gene in the innate immune response, the copepods were exposed to lipopolysaccharide (LPS) and two Vibrio sp. After exposure to different concentrations of LPS and Vibrio sp., T. japonicus NOS transcription was significantly increased over time in a dose-dependent manner, and the NO/nitrite concentration increased as well. Taken together, our findings suggest that T. japonicus NOS transcription is induced in response to an immune challenge as part of the conserved innate immunity.

Cellular, biochemical, and molecular characterization of nitric oxide synthase expressed in the nervous system of the prosobranch Stramonita haemastoma (Gastropoda, Neogastropoda)

Nitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca(2+) /CaM-dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L-arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane-bound. Molecular cloning of the full-length Stramonita NOS (Sh-NOS) by reverse-transcription polymerase chain reaction (RT-PCR) followed by 5' and 3' RACE showed that Sh-NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N-terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh-NOS gene was evaluated by RT-PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods.

Distribution and characterization of nitric oxide synthase in the nervous system of Triatoma infestans (Insecta: Heteroptera)

The biochemical characterization of nitric oxide synthase (NOS) and its distribution in the central nervous system (CNS) were studied in the heteropteran bug Triatoma infestans. NOS-like immunoreactivity was found in the brain, subesophageal ganglion, and thoracic ganglia by using immunocytochemistry. In the protocerebrum, NOS-immunoreactive (IR) somata were detected in the anterior, lateral, and posterior soma rinds. In the optic lobe, numerous immunostained somata were observed at the level of the first optic chiasma, around the lobula, and in the proximal optic lobe. In the deutocerebrum, NOS-IR perikarya were mainly observed in the lateral soma rind, surrounding the sensory glomeruli, and a few cell bodies were seen in association with the antennal mechanosensory and motor neuropil. No immunostaining could be detected in the antennal nerve. The subesophageal and prothoracic ganglia contained scattered immunostained cell bodies. NOS-IR somata were present in all the neuromeres of the posterior ganglion. Western blotting showed that a universal NOS antiserum recognized a band at 134 kDa, in agreement with the expected molecular weight of the protein. Analysis of the kinetics of nitric oxide production revealed a fully active enzyme in tissue samples of the CNS of T. infestans.

Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2′,5′-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS–PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K m value of 13.4×10 −6 M for L-arginine and V max of 35.3 nmol min −1 mg −1 protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca 2+ were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.

Identification and characterization of nitric oxide synthase in Salmonella typhimurium.

The presence of the nitric oxide synthase (NOS) enzyme from Salmonella typhimurium (S. typhimurium) was identified by measuring radiolabeled L-[3H]citrulline and NO, and Western blot analysis. NOS was partially purified by both Mono Q ion exchange and Superose 12HR size exclusion column chromatography, sequentially. The molecular weight of NOS was estimated to be 93.3 kDa by Western blot analysis. The enzyme showed a significant dependency on the typical NOS cofactors; an apparent Km for L-arginine of 34.7 mM and maximum activity between 37 degrees C and 43 degrees C. The activity was inhibited by NOS inhibitors such as aminoguanidine and N(G),N(G)-dimethyl-L-arginine. Taken together, partially purified NOS in S. typhimurium is assumed to be a different isoform of mammalian NOSs.

Characterization of nitric oxide synthase in the rat parotid gland

Nitric oxide (NO) acts as an inter- and intracellular signalling molecule of various cells such as vascular endothelium, macrophages, and neurones. NO is produced by nitric oxide synthase (NOS) from l-arginine. Here the characteristics of NOS in the rat parotid gland were investigated. Approximately 74% of total activity of NOS was present in the cytosolic fraction. For full activation of the NOS in the cytosolic fraction, tetrahydroxybiopterin, NADPH, Ca 2+ and calmodulin were needed as cofactors, because the activity was clearly reduced in the absence of tetrahydroxybiopterin, NADPH, or Ca 2+, or in the absence of calmodulin and presence of trifluoperazine, a calmodulin antagonist, in the reaction mixture. The partially purified NOS activity was completely abolished in the absence of calmodulin or Ca 2+, and activated by them in a dose-dependent manner; EC 50 for calmodulin and Ca 2+ were 10 and 340 nM, respectively. The K m for l-arginine was 1.57 μM. Immunoblot analysis revealed that a 165-kDa protein band in the rat parotid gland cytosolic fraction cross-reacted with a rabbit polyclonal antibody against human brain NOS. These results suggest that NOS of the rat parotid gland is a neuronal isoform and that its activity is regulated by physiological concentrations of calmodulin and Ca 2+.

Biochemical and functional characterization of nitric oxide synthase III gene transfer using a replication-deficient adenoviral vector

Nitric oxide (NO) produced in endothelial cells has been implicated in the regulation of blood pressure, regional blood flow, inhibition of platelet aggregation, and endothelial and vascular smooth muscle cell proliferation. In a variety of cardiovascular disease states, such as atherosclerosis, arterial hypertension, and restenosis, expression of endothelial NO synthase (NOS-III) and endothelial NO production appear to be altered. Thus, NOS-III is an attractive target for cardiovascular gene therapy for which adenoviral vectors are one of the most effective vector systems. Therefore, a recombinant adenoviral vector expressing NOS-III (adenovirus type 5 [Ad5] cytomegalovirus [CMV] NOSIII) was constructed and biochemically and pharmacologically characterized both in vitro and in intact cells. Ad5CMVNOSIII-derived recombinant NOS-III was successfully expressed, as shown by immunoprecipitation and immunocytochemistry, and biologically active, as shown by functional assays in human primary umbilical vein and EA.hy926 endothelial cells, as well as 293 human embryonic kidney and Chinese hamster ovary cells. The K m values for NADPH and l-arginine and the K a for tetrahydrobiopterin as well as the enzyme’s dependency on other cofactors were similar to recombinant reference enzyme and literature values. NOS-III expression levels correlated linearly with the multiplicity of infection with Ad5CMVNOSIII and lasted for at least 8 days. NOS-III transfection inhibited endothelial cell proliferation. In conclusion, adenovirus-mediated gene transfer of Ad5CMVNOSIII to vascular and non-vascular cells resulted in the dose-dependent expression of intact, physiologically regulated, and functionally active NOS-III.

Functional and molecular characterization of nitric oxide synthase in the endometrium and myometrium of pregnant sheep during the last third of gestation

Objective: This study was undertaken to characterize the biochemical and expression profiles of the nitric oxide synthase isoforms present in the sheep uterus during late gestation. Study Design: Myometrium and endometrium were obtained from 28 time-mated pregnant sheep that were under halothane general anesthesia. Tissues were kept frozen at –80°C until they were homogenized for the measurement of (1) nitric oxide synthase activity according to the carbon 14–labeled arginine-citrulline conversion assay, (2) nitric oxide synthase protein mass according to Western blot analysis, and (3) nitric oxide synthase messenger ribonucleic acid according to the ribonuclease protection assay. The nitric oxide synthase activity assay included 8 parallel treatments for biochemical characterization, in particular with the arginase inhibitors ornithine and (+)-S-2-amino-5-iodoacetamidopentanoic acid. Results: The biochemical characterization of nitric oxide synthase indicated that the predominant form of nitric oxide synthase in endometrium and myometrium (80%-90%) was calcium-calmodulin dependent. In endometrium 50% of reduced nicotinamide adenine dinucleotide–dependent arginine metabolism was accounted for by the presence of alternative arginine metabolic pathways. Expressions of type 1 and type 3 nitric oxide synthase were demonstrated in endometrium and myometrium by Western blot and ribonuclease protection assay. Although no significant decrease in nitric oxide synthase activity or protein mass was observed, a significant decrease in myometrial type 1 nitric oxide synthase messenger ribonucleic acid occurred in sheep not in labor at 140 days’ gestation ( P < .05 by analysis of variance; term is 144 ± 5 days). Conclusion: In the gravid sheep uterus the predominant nitric oxide synthase isoforms are type 1 in myometrium and type 3 in endometrium. Despite a decrease in type 1 nitric oxide synthase messenger ribonucleic acid, enzymatic activity and type 1 nitric oxide synthase protein mass do not decrease before parturition. (Am J Obstet Gynecol 1999;181:116-25.)

Partial biochemical characterization of nitric oxide synthase in the caudal spinal cord of the teleost Oreochromis niloticus

The present study demonstrates that a NADPH/Ca 2+-dependent nitric oxide synthase (NOS) activity is present in the soluble and in the particulate fractions of fish caudal spinal cord homogenates, both activities being inhibited by calmodulin inhibitors (W7 and/or TFP) and by the NOS inhibitor l-NAME. Moreover, Western blot analysis using either anti-NOS I or anti-NOS III antibodies shows that the soluble enzyme corresponds to the brain NOS (NOS-I) of mammals, whereas the particulate one is likely attributable to NOS I and/or NOS III (ecNOS) enzymes. To confirm the nitric oxide (NO) production by the caudal spinal cord homogenates, the NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored spectroscopically. The present results are consistent with a constitutive, Ca 2+-calmodulin-dependent, NO production by the caudal neurosecretory system.

Localization and characterization of nitric oxide synthase at the frog neuromuscular junction.

The frog neuromuscular junction is sensitive to nitric oxide (NO), since exogenously applied NO reduces the release of transmitter b presynaptic terminals and the size of ATP-induced Caoffresponses in perisynaptic Schwann cells. This study aimed at determining whether an NO synthase (NOS) is present at the neuromuscular junction, notably in perisynaptic Schwann cells, the glial cells at this synapse. The NADPH-diaphorase (NADPH-d) histochemical technique revealed the presence of NOS in cell bodies and presumed processes of perisynaptic Schwann cells. Incubation with NOS inhibitors, NG-nitro-L-arginine methyl ester or NG-nitro-L-arginine-acetate abolished the NADPH-d staining. Moreover, L-arginine, the precursor of NO, impeded the blockade by NOS inhibitors, establishing the NOS specificity of NADPH-d staining in frog tissue. The pattern of labelling with a polyclonal antibody against the neuronal form of NOS was similar to the NADPH-d staining, also suggesting the presence of a neuronal NOS in perisynaptic Schwann cells. Using electron microscopy the NOS immunostaining was found at the membrane and occasionally in the cytoplasm of perisynaptic Schwann cells and was not detected in the nerve terminal or muscle. There was no enzymatic or immunocytochemical labelling of NOS 6 days after denervation. It is concluded that NOS is present in frog perisynaptic Schwann cells. The presence of this endogenous NOS suggests that NO may act as a diffusible glial messenger to modulate synaptic activity and synapse formation at the neuromuscular junction.

Photic control of nitric oxide synthase activity in golden hamster retina.

A characterization of nitric oxide synthase (NOS) in the golden hamster retina was performed. Enzymatic activity was partially Ca2+ and calmodulin-dependent, required NADPH, and was inhibited by L-arginine analogs. Retinal NOS activity was higher at midnight than at midday. When the hamster were placed under constant darkness for 24 or 48 h, and killed at equivalent time points, representing subjective day and subjective night respectively, the differences in NOS activity disappeared. One hour of darkness during the day increased, while the same period of light during the night decreased, retinal NOS activity. From these results, it might be presumed that hamster retinal NOS activity is regulated by the photic stimulus.

Characterization of Nitric Oxide Synthase in the Cerebellum of the Chicken

Nitric oxide synthase (NOS) activity in the chicken brain was examined, and higher activity was detected in the cerebellum. The NOS activity in the cerebellum was significantly inhibited in vitro by W-5, W-7 and trifluoperazine, and recovery of the activity was observed by the addition of calmodulin, suggesting that NOS is calmodulin dependent. Moreover, effects of EGTA and Ca2+ on NOS activity were also examined, and the results indicated that the enzyme is Ca2+ dependent. In addition, cofactors such as NADPH, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were required for the full expression of NOS. The results suggest that chicken cerebellum NOS has similar properties to those of mammalian neural NOS.

Localization and characterization of nitric oxide synthase in the rat suprachiasmatic nucleus: evidence for a nitrergic plexus in the biological clock.

Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH-diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of L-[3H]arginine to L-[3H]-citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca2+/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.

Characterization of nitric oxide synthase (NOS) expression in the urinary bladder of the ovariectomized (OVX) sheep and the effect of estrogen treatment.

Characterization of nitric oxide synthase (NOS) expression in the urinary bladder of the ovariectomized (OVX) sheep and the effect of estrogen treatment.

Purification and characterization of nitric oxide synthase (NOSNoc) from a Nocardia species

We previously reported on the occurrence, partial purification, and preliminary characterization of the first reported bacterial nitric oxide synthase. The soluble Nocardia enzyme, designated NOSNoc, has now been purified 1,353-fold by a combination of 2',5'-ADP-agarose affinity chromatography and hydroxylapatite chromatography. NOSNoc runs as a band of M(r) 51,900 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 110.6 +/- 0.5 kDa by gel filtration, indicating that the native enzyme exists as a homodimer in solution. An N-terminal 15-amino-acid sequence was determined for NOSNoc, showing it to be different from known mammalian NOSs. NG-Hydroxy-L-arginine was confirmed to be an intermediate in the enzymatic reaction by stoichiometric determinations of oxygen uptake, NADPH oxidation, NO formation as measured by nitrite determinations, citrulline formation, and kinetic studies. NOSNoc was competitively inhibited by NG-methyl- and NG-nitro-L-arginine with either L-arginine or NG-hydroxyl-L-arginine as the substrate. Furthermore, the stability and pH and temperature optima of NOSNoc have been established.

Poster 3: Immunohistochemical Characterization of Nitric Oxide Synthase in Squamous Cell Carcinoma of the Head and Neck

Poster 3: Immunohistochemical Characterization of Nitric Oxide Synthase in Squamous Cell Carcinoma of the Head and Neck

Detection and characterization of nitric oxide synthase in the mammalian cochlea

The messenger molecule nitric oxide (NO) is involved in blood flow regulation, cytotoxicity, and neural signalling, processes that are important in the physiology and pathophysiology of the mammalian cochlea. However, neither the presence of NO nor its synthetic enzyme, NO synthase, has been established in the peripheral auditory system. NO synthase activity, measured as the enzymatic conversion of radioactive arginine to citrulline, was predominantly soluble in the auditory nerve, lateral wall, vetibule and cochlear neuroepithelium. N-methyl- l-arginine and trifluoperazine inhibited NO synthase activity in the lateral wall and auditory nerve. Histochemical staining by NADPH-diaphorase localized NOS activity to the lateral wall and the neuronal elements of the organ of Corti. Based on these results, the predominant NO synthase isoform in the cochlea is the neuronal type-I isoform.

Characterization of nitric oxide synthase in the opossum esophagus

Background/Aims: Nitric oxide mediates the nonadrenergic, noncholinergic neural control of esophageal motor function. The purpose of this study was to characterize the NO synthase found in the muscularis propria of the opossum esophagus and determine its distribution along the esophagus. Methods: Esophageal muscle was homogenized in HEPES buffer and ultracentrifuged. The supernatant was exposed to [3H]l-arginine. The [3H]l-citrulline produced by NO synthase was separated from [3H]l-arginine with a Dowex AG 50 W-X8 column (Biorad, Hercules, CA). Assays were performed in the presence and absence of Ca2+ or reduced nicotinamide adenine dinucleotide phosphate (NADPH). The distribution of NO synthase activity along the esophagus was determined. Results: The apparent Michaelis constant and maximum velocity of NO synthase were 7.5 ± 1.4 μmol/L l-arginine and 76.0 ± 17.3 pmol·mg protein−1 ·min−1, respectively. The enzyme required both Ca2+ and NADPH for activity. Smooth muscle tissue from the lower esophageal sphincter and the esophageal body 1–2 cm or 5–6 cm above the lower esophageal sphincter differed little in enzymatic activity, ranging from 0.97 to 1.27 pmol·mg wet wt−1·min−1. Striated muscle had less activity with 0.40 pmol·mg wet wt−1·min−1. Conclusions: These data indicate the presence of a constitutive NO synthase in the esophagus of the opossum.

Subcellular localization and characterization of nitric oxide synthase(s) in endothelial cells: physiological implications.

Endothelial cells (EC) contain a constitutive Ca2+/calmodulin-dependent nitric oxide (NO) synthase (cNOS) which plays an important role in the local control of vascular tone. We compared the subcellular distribution of this enzyme in cultured and freshly isolated pig EC by determination of specific cNOS activity and immunoblot analysis. Similar studies were also performed with cultured and freshly isolated bovine and cultured human EC. Enzyme activity was predominantly (> 70%) associated with the particulate fraction of all EC types tested and was highest in freshly isolated porcine EC. Both specific cNOS activity and immunoreactivity were substantially higher (> 3-fold) in the microsomal as compared with the soluble fraction of all EC types tested. In freshly isolated pig EC, these two fractions also differed in terms of their Ca(2+)-dependency, pH optimum and inhibitor specificity. EC may thus contain either two different cNOS isoenzymes or a single enzyme, the conformation of which differs between the soluble and membrane-bound state. Moreover, detailed subcellular fractionation of freshly isolated pig EC revealed that the distribution of cNOS activity closely resembled that of the plasma membrane marker 5'-nucleotidase, suggesting that most, if not all, of the cNOS activity in these cells is associated with the plasma membrane. This localization might render the enzyme more susceptible to activation by physical stimuli, such as a shear stress-induced change in the fluidity of the plasma membrane. Moreover, the continuous exposure to shear stress in vivo may also upregulate cNOS expression in EC, since specific enzyme activity, immunoreactivity and basal NO release were significantly higher in freshly isolated EC as compared with cultured EC.