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P Sarti

Publications and source records attributed to P Sarti.

At least 19 recordsLinked to original sources
PubMed2004

Trichomonas vaginalis degrades nitric oxide and expresses a flavorubredoxin-like protein: a new pathogenic mechanism?

Besides possessing many physiological roles, nitric oxide (NO) produced by the immune system in infectious diseases has antimicrobial effects. Trichomoniasis, the most widespread non-viral sexually transmitted disease caused by the microaerophilic protist Trichomonas vaginalis, often evolves into a chronic infection, with the parasite able to survive in the microaerobic, NO-enriched vaginal environment. We relate this property to the finding that T. vaginalis degrades NO under anaerobic conditions, as assessed amperometrically. This activity, which is maximal (133 +/- 41 nmol NO/10(8) cells per minute at 20 degrees C) at low NO concentrations (< or = 1.2 microM), was found to be: (i) NADH dependent, (ii) cyanide insensitive and (iii) inhibited by O(2). These features are consistent with those of the Escherichia coli A-type flavoprotein (ATF), recently discovered to be endowed with NO reductase activity. Using antibodies against the ATF from E. coli, a protein band was immunodetected in the parasite grown in a standard medium. If confirmed, the expression of an ATF in eukaryotes suggests that the genes coding for ATFs were transferred during evolution from anaerobic Prokarya to pathogenic protists, to increase their fitness for the microaerobic, parasitic life style. Thus the demonstration of an ATF in T. vaginalis would appear relevant to both pathology and evolutionary biology. Interestingly, genomic analysis has recently demonstrated that Giardia intestinalis and other pathogenic protists have genes coding for ATFs.

Animals↗
PubMed2004

Morphine but not fentanyl and methadone affects mitochondrial membrane potential by inducing nitric oxide release in glioma cells.

We have observed that treatment of human glioma cells with morphine in the nanomolar range of concentration affects the mitochondrial membrane potential. The effect is specific to morphine and is mediated by naloxone-sensitive receptors, and is thus better observed on glioma cells treated with desipramine; moreover, the mitochondrial impairment is not inducible by fentanyl or methadone treatment and is prevented by the nitric oxide (NO) synthase inhibitor L-NAME. We conclude that in cultured glioma cells, the morphine-induced NO release decreases the mitochondrial membrane potential, as one might expect based on the rapid inhibition of the respiratory chain by NO. The identification of new intra-cellular pathways involved in the mechanism of action of morphine opens additional hypotheses, providing a novel rationale relevant to the therapy and toxicology of opioids.

Adjuvants, Anesthesia↗
PubMed2003

Control of respiration by nitric oxide in Keilin-Hartree particles, mitochondria and SH-SY5Y neuroblastoma cells.

The pattern of cytochrome c oxidase inhibition by nitric oxide (NO) was investigated polarographically using Keilin-Hartree particles, mitochondria and human neuroblastoma cells. NO reacts with purified cytochrome c oxidase forming either a nitrosyl- or a nitrite-inhibited derivative, displaying distinct kinetics and light sensitivity of respiration recovery in the absence of free NO. Keilin-Hartree particles or cells, respiring either on endogenous substrates alone or in the presence of ascorbate, as well as state 3 and state 4 mitochondria respiring on glutamate and malate, displayed the rapid recovery characteristic of the nitrite derivative. All systems, when respiring in the presence of tetramethyl-p-phenylenediamine, were characterised by the slower, light-sensitive recovery typical of the nitrosyl derivative. Together the results suggest that the reaction of NO with cytochrome c oxidase in situ follows two alternative inhibition pathways, depending on the electron flux through the respiratory chain.

Animals↗
PubMed2001

The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity.

The cytochrome cbb3 is an isoenzyme in the family of cytochrome c oxidases. This protein purified from Pseudomonas stutzeri displays a cyanide-sensitive nitric oxide reductase activity (Vmax=100+/-9 mol NO x mol cbb3(-1) x min(-1) and Km=12+/-2.5 microm), which is lost upon denaturation. This enzyme is only partially reduced by ascorbate, and readily re-oxidized by NO under anaerobic conditions at a rate consistent with the turnover number for NO consumption. As shown by transient spectroscopy experiments and singular value decomposition (SVD) analysis, these results suggest that the cbb3-type cytochromes, sharing structural features with bacterial nitric oxide reductases, are the enzymes retaining the highest NO reductase activity within the heme-copper oxidase superfamily.

Electron Transport Complex IV↗
PubMed2000

Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects.

The reactions of nitric oxide (NO) with the turnover intermediates of cytochrome c oxidase were investigated by combining amperometric and spectroscopic techniques. We show that the complex of nitrite with the oxidized enzyme (O) is obtained by reaction of both the "peroxy" (P) and "ferryl" (F) intermediates with stoichiometric NO, following a common reaction pathway consistent with P being an oxo-ferryl adduct. Similarly to chloride-free O, NO reacted with P and F more slowly [k approximately (2-8) x 10(4) M(-1) s(-1)] than with the reduced enzyme (k approximately 1 x 10(8) M(-1) s(-1)). Recovery of activity of the nitrite-inhibited oxidase, either during turnover or after a reduction-oxygenation cycle, was much more rapid than nitrite dissociation from the fully oxidized enzyme (t(1/2) approximately 80 min). The anaerobic reduction of nitrite-inhibited oxidase produced the fully reduced but uncomplexed enzyme, suggesting that reversal of inhibition occurs in turnover via nitrite dissociation from the cytochrome a(3)-Cu(B) site: this finding supports the hypothesis that oxidase may have a physiological role in the degradation of NO into nitrite. Kinetic simulations suggest that the probability for NO to be transformed into nitrite is greater at low electron flux through oxidase, while at high flux the fully reduced (photosensitive) NO-bound oxidase is formed; this is fully consistent with our recent finding that light releases the inhibition of oxidase by NO only at higher reductant pressure [Sarti, P., et al. (2000) Biochem. Biophys. Res. Commun. 274, 183].

Animals↗
PubMed2000

Nitric oxide and cytochrome c oxidase: mechanisms of inhibition and NO degradation.

NO inhibits mitochondrial respiration by reacting with either the reduced or the oxidized binuclear site of cytochrome c oxidase, leading respectively to accumulation of cytochrome a(2+)(3)-NO or cytochrome a(3+)(3)-NO(-)(2) species. Exploiting the unique light sensitivity of the cytochrome a(2+)(3)-NO, we show that under turnover conditions, depending on the cytochrome c(2+) concentration, either the cytochrome a(2+)(3)-NO or the nitrite-bound enzyme is formed. The predominance of one of the two inhibitory pathways depends on the occupancy of the turnover intermediates. In the dark, the respiration recovers at the rate of NO dissociation (k' = 0.01 s(-1) at 37 degrees C). Illumination of the sample speeds up recovery rate only at higher reductant concentrations, indicating that the inhibited species is cytochrome a(2+)(3)-NO. When the reaction occurs with the oxidized binuclear site, light has no effect and NO is oxidized to harmless nitrite eventually released in the bulk, accounting for catalytic NO degradation.

Animals↗
PubMed2000

The ratio between the fast and slow forms of bovine cytochrome c oxidase is changed by cholate or nucleotides bound to the cholate-binding site close to the cytochrome a3/CuB binuclear centre.

We determined the fraction of 'slow' and 'fast' conformations of bovine cytochrome c oxidase, following the kinetics of cyanide binding to the oxidized enzyme. We investigated whether treatment of heart mitochondrial particles with different commercially available types of cholate (standard and ultrapure) can affect the fraction of cytochrome c oxidase in the two states. Compared to standard cholate, the use of ultra-pure cholate for solubilization of heart mitochondrial particles significantly increased the fraction of the fast enzyme. Complete homogeneity (approximately 100% fast) was observed when cytochrome c oxidase was solubilized with ultra-pure cholate from heart mitochondrial particles pre-equilibrated with AMP; equilibration with ADP yielded a much smaller fraction of fast enzyme (approximately 35%). These observations are discussed on the basis of the structural relationships between the known cholate-binding site and the binuclear cytochrome a3-CuB site: variation in the occupancy of this binding site with cholate or nucleotides may modify reactivity of the oxidized binuclear centre towards cyanide.

Adenosine Diphosphate↗
PubMed1999

The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications.

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba(3) and caa(3)) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) under reducing anaerobic conditions. The rate of NO consumption and N(2)O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 +/- 0.7 mol NO/mol ba(3) x min and 32 +/- 8 mol NO/mol caa(3) x min at [NO] approximately 50 microM and 20 degrees C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme x min), is definitely significant. We also show that for ba(3) oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar Cu(B)(+) coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb(3) terminal oxidases. Our findings represent functional evidence in support of this hypothesis.

Copper↗
PubMed1999

Nitric oxide and cellular respiration.

The role of nitric oxide (NO) as a signalling molecule involved in many pathophysiological processes (e.g., smooth muscle relaxation, inflammation, neurotransmission, apoptosis) has been elaborated during the last decade. Since NO has also been found to inhibit cellular respiration, we review here the available information on the interactions of NO with cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. The effect of NO on cellular respiration is first summarized to present essential evidence for the fact that NO is a potent reversible inhibitor of in vivo O2 consumption. This information is then correlated with available experimental evidence on the reactions of NO with purified COX. Finally, since COX has been proposed to catalyze the degradation of NO into either nitrous oxide (N2O) or nitrite, we consider the putative role of this enzyme in the catabolism of NO in vivo.

Animals↗
PubMed1999

Mechanism of S-nitrosothiol formation and degradation mediated by copper ions.

Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu(2+), but not Fe(3+), induces in the presence of NO a fast S-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu(+) is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu(2+), the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu(2+) in S-nitrosothiol formation with the known destabilizing effect of Cu(+), providing a unique simple picture where the redox state of copper determines either the NO release from S-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation.

Animals↗
PubMed1999

Permeability of rat heart myocytes to cytochrome c.

Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellular vacuoles and plasma membrane blebbing. The damaged myocytes displayed impaired N,N'-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respiratory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluorescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 microM cytochrome c, establishing the threshold below which the turnover rate by cytochrome c oxidase in the cell depends on cytochrome concentration. Mildly damaged cardiac myocytes, as indicated by cell shortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeable to fluorescein-conjugated cytochrome c. The results, while consistent with an early cytochrome c release observed at the beginning of cell death, elucidate the role played by cytochrome c in the kinetic control of mitochondrial electron transfer under pathological conditions, particularly those involving the terminal part of the respiratory chain. These data are the first to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c.

Animals↗
PubMed1999

Effect of intravenous clarithromycin on interdigestive gastroduodenal motility of patients with functional dyspepsia and Helicobacter pylori gastritis.

Gastroduodenal motility of 16 patients complaining of functional dyspepsia and Helicobacter pylori gastritis was recorded by means of a low-compliance manometric system with four recording ports in the stomach and four in the duodenum. Clarithromycin (CLA) 250 mg (group A: 8 patients) or normal saline solution (group B: 8 patients) was infused intravenously randomly and in double-blind manner 30 min after the end of the first recorded activity front (AF) of the migrating motor complex or, in the absence of AFs, after 200 min of recording, continuing the recording until an AF was observed during the subsequent 200 min. CLA administration was followed by a typical gastroduodenal AF in a significantly higher number of patients than saline administration. In addition, the time-lag between the drug administration and the appearance of AFs was 22 min +/-7.4 (mean +/- SD), significantly shorter than after saline (109+/-56 min) and the CLA-related duodenal AFs showed a duration of 7.4 min +/-1.6 in group A, significantly longer than that of the spontaneous AFs (3.5 min +/-1), while in group B AF duration after saline was not significantly different from that of the spontaneous ones. In conclusion, clarithromycin is able to stimulate cyclic interdigestive gastroduodenal motility. This prokinetic property of clarithromycin is not unexpected because it is a macrolide like erythromycin, the prokinetic activity of which is well known, and could be utilized for therapeutic uses.

Adolescent↗
PubMed1999

Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy.

With the electro-driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer-aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady-state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic-to-measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20-fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.

Fluorescence↗
PubMed1998

Chloride bound to oxidized cytochrome c oxidase controls the reaction with nitric oxide.

The reaction of nitric oxide (NO) with oxidized fast cytochrome c oxidase was investigated by stopped-flow, amperometry, and EPR, using the enzyme as prepared or after "pulsing." A rapid reduction of cytochrome a is observed with the pulsed, but not with the enzyme as prepared. The reactive species (lambdamax = 424 nm) reacts with NO at k = 2.2 x 10(5) M-1 s-1 at 20 degreesC and is stable for hours unless Cl- is added, in which case it decays slowly (t1/2 approximately 70 min) to an unreactive state (lambdamax = 423 nm) similar to the enzyme as prepared. Thus, Cl- binding prevents a rapid reaction of NO with the oxidized binuclear center. EPR experiments show no new signals within 15 s after addition of NO to the enzyme as prepared. Amperometric measurements show that the pulsed NO-reactive enzyme reacts with high affinity and a stoichiometry of 1 NO/aa3, whereas the enzyme as prepared reacts to a very small extent (<20%). In both cases, the reactivity is abolished by pre-incubation with cyanide. These experiments suggest that the effect of "pulsing" the enzyme, which leads to enhanced NO reactivity, arises from removing Cl- bound at the oxidized cytochrome a3-CuB site.

Animals↗
PubMed1998

Intermediates in the catalytic cycle of lentil (Lens esculenta) seedling copper-containing amine oxidase.

Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. gamma-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.

Amine Oxidase (Copper-Containing)↗
PubMed1998

Cytochrome c oxidase does not catalyze the anaerobic reduction of NO.

A possible role of reduced cytochrome c oxidase in the metabolism of nitric oxide (NO) has been examined with amperometric and stopped-flow photometric techniques. Reduced purified cytochrome c oxidase and mitochondria showed no catalytic reaction with NO under anaerobic conditions within more than 30 minutes. Only fast binding of NO to the reduced enzyme in a 1:1 stoichiometric ratio was observed. The NO binding rate was strongly decreased in the presence of 1 mM cyanide. These data indicate that, contrary to previous proposals, cytochrome c oxidase in the absence of oxygen does not contribute to physiological NO metabolism.

Anaerobiosis↗