Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y₂ receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP₃ receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca²⁺-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y₂ receptors. These results show that P2Y₂ receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP₃/intracellular Ca²⁺ release and Ca²⁺ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.     

Rapamycin sensitive mTOR activation mediates nerve growth factor (NGF) induced cell migration and pro-survival effects against hydrogen peroxide in retinal pigment epithelial cells

Patients with age related macular degeneration (AMD) have a loss of vision in the center of the visual field. Oxidative stress plays an important role in this progress. Nerve growth factor (NGF) is important for the survival and maintenance of sympathetic and sensory neurons and NGF eye drops improve visual acuity and electro-functional activity in patients with AMD. However, the molecular mechanisms and signaling events involved in this have not been fully investigated. Using cultured human retinal pigment epithelial (RPE) cells, we demonstrate here that NGF protects RPE cells against hydrogen peroxide (H₂O₂)-induced cell apoptosis. NGF also induces RPE cell migration, the latter is important for retinal regeneration and the recovery from AMD. H₂O₂ decreases S6 phosphorylation and cell viability, which is restored by NGF. Rapamycin, the pharmacologic inhibitor of mammalian target of rapamycin (mTOR), diminished NGF-induced S6 phosphorylation, cell migration and protective effects against oxidative stress. Collectively, we conclude that activation of rapamycin sensitive mTOR signaling mediates NGF induced cell migration and pro-survival effects in H₂O₂ treated RPE cells.     

P2Y receptors on astrocytes and microglia mediate opposite effects in astroglial proliferation

Nucleotides released upon brain injury signal to astrocytes and microglia playing an important role in astrogliosis, but the participation of microglia in the purinergic modulation of astrogliosis is still unclear. Highly enriched astroglial cultures and co-cultures of astrocytes and microglia were used to investigate the influence of microglia in the modulation of astroglial proliferation mediated by nucleotides. In highly enriched astroglial cultures, adenosine-5’-triphosphate (ATP), adenosine 5’-O-(3-thio)-triphosphate (ATPγS), adenosine 5’-O-(3-thio)-diphosphate (ADPβS; 0.01–1 mM), and adenosine-5’-diphosphate (ADP; 0.1–1 mM) increased proliferation up to 382%, an effect abolished in co-cultures containing 8% of microglia. The loss of ATP proliferative effect in co-cultures is supported by its fast metabolism and reduced ADP accumulation, an agonist of P2Y_1,12 receptors that mediate astroglial proliferation. No differences in ADPβS and ATPγS metabolism or P2Y_1,12 receptors expression were found in co-cultures that could explain the loss of their proliferative effect. However, conditioned medium from microglia cultures or co-cultures treated with ADPβS, when tested in highly enriched astroglial cultures, also prevented ADPβS proliferative effect. None of the uracil nucleotides tested had any effect in proliferation of highly enriched astroglial cultures, but uridine-5′-triphosphate (UTP; 0.1–1 mM) inhibited proliferation up to 66% in co-cultures, an effect that was dependent on uridine-5’-diphosphate (UDP) accumulation, coincident with a co-localization of P2Y₆ receptors in microglia and due to cell apoptosis. The results indicate that microglia control astroglial proliferation by preventing the proliferative response to adenine nucleotides and favouring an inhibitory effect of UTP/UDP. Several microglial P2Y receptors may be involved by inducing the release of messengers that restrain astrogliosis, a beneficial effect for neuronal repair mechanisms following brain injury.     

Cationic phospholiposomes: efficient delivery vehicles of anticancer derivatives of ATP to multiple myeloma cells

Analogs of adenosine triphosphate (ATP) with substitutions at the 8-position have been shown to be cytotoxic to multiple myeloma, one of the most prevalent and serious blood cancers. However, these drugs do not readily cross biological membranes and are very sensitive to phosphatases present in body fluids. To circumvent these disadvantages, 8-substituted ATPs were encapsulated into cationic phospholiposomes generated from cationic phosphatidylcholines (EDOPC; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, and EDPPC, the corresponding dipalmitoyl homolog), compounds with low toxicity that readily form liposomes. Vortexing was an efficient encapsulation procedure, more so than freeze-thawing. At the lipid:drug ratio of 5:1 (mol/mol), 20% of 8-Br-ATP was encapsulated within EDOPC liposomes. Efficient encapsulation and retention of 8-NH₂-ATP required the inclusion of cholesterol. Liposomes of EDOPC:cholesterol (55:45 mole/mole), at a lipid:drug mole ratio of 10:1, captured ~40% of the drug presented. Cytotoxicity assays of this formulation on multiple myeloma cells in culture showed encapsulated drug to be up to 10-fold more effective than free drug, depending upon dose. Intracellular distribution studies (based on fluorescent derivatives of lipids and of ATP) revealed that both liposomes and drug were taken up by multiple myeloma cells, and that uptake of a fluorescent ATP derivative was significantly greater when encapsulated than when free. Liposomes prepared from EDPPC, having a higher phase-transition temperature than EDOPC, captured 8-NH₂-ATP satisfactorily and released it more slowly than the unsaturated formulations, but were also less cytotoxic. The superior encapsulation efficiencies of the positively charged liposomes can be understood in terms of the electrostatic double layer due to a very high positive charge density on their inner surface. Electrostatic augmentation of encapsulation for small vesicles can be dramatic, easily exceeding an order of magnitude.     

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y₂ or P2Y₄ receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y₂ or P2Y₄ receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.     

γ-Irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells

Background

Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.

Methods

Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X₇ receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X₇ receptor.

Results

Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X₇ receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X₇ receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X₇ receptor channel plays a significant role in mediating the ATP release.

General Significance

We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.     

Isolation and measurement of urinary 8-iso-prostaglandin F2α by high-performance liquid chromatography and gas chromatography–mass spectrometry

8-iso-Prostaglandin F_2α (8-iso-PGF_2α) is a product of free radical-catalyzed peroxidation of arachidonic acid. Measurement of its urinary excretion has been proposed as an index of oxidative status in vivo. A stable isotope dilution method for its quantification by gas chromatography–electron capture chemical ionization mass spectrometry is described. Sample cleanup required the combined use of high-performance liquid chromatography and thin-layer chromatography. The inter-assay R.S.D. in two separate determinations was 1.6 (n=4) and 2.3% (n=4). The accuracy of the assay was evaluated through recovery experiments. The equation of the regression plot correlating the amounts added and recovered was y=0.91x−0.31, r=0.9916 (n=12). The pair of fragment ions ([M−181]⁻) at m/z 569 and m/z 573 was monitored for quantification. The mean 8-iso-PGF_2α excretion rate was 528±127 (S.D.) ng per day in five male volunteers and 730±305 ng per day in six females. Intake of 80 mg of lycopene per day by eleven volunteers for four weeks resulted in a non-significant reduction of 8-iso-PGF_2α excretion.     

Endothelium-dependent epithelial–mesenchymal transition of tumor cells: Exclusive roles of transforming growth factor β1 and β2

Background

Induction of epithelial–mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not.

Methods

Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor β (TGFβ) antibodies were used to neutralize TGFβ₁, TGFβ₂ and TGFβ₃. Expression and release of TGFβ proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively.

Results

Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFβ₁ and TGFβ₂ proteins and much lower level of TGFβ₃ protein. Conditioned medium from these endothelial cells contained TGFβ₁ and TGFβ₂, but TGFβ₃ could not be detected. Neutralizing antibody against each of TGFβ₁ or TGFβ₂ did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFβ₁ and TGFβ₂ completely abolished it.

Conclusions

Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFβ₁ and TGFβ₂.

General significance

The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium.     

Iron transport by proteoliposomes containing mitochondrial F1Fo ATP synthase isolated from rat heart

In this work, we present evidence of Fe²⁺ transport by rat heart mitochondrial F₁F_o ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 °C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the α, β, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg⁺², Ca⁺², Mn⁺², Zn⁺², Cu⁺², Fe⁺³, and Al⁺³ was observed. Moreover, Cu⁺², even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H⁺.     

Covalent Binding of 1,N 6-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP synthase

Catalytic and noncatalytic sites of the chloroplast coupling factor (CF₁) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N⁶-ethenoadenosine diphosphate. The modified CF₁ was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N⁶-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N⁶-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N⁶-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N⁶-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed.     

Photoautotrophic growth of sugarcane plantlets <Emphasis Type="Italic">in vitro</Emphasis> as affected by photosynthetic photon flux and vessel air exchanges

Summary Explants of sugarcane, a C₄ plant, were cultured in vitro for 18d on Floridalite (a solid cube consisting of vermiculite and cellulose fibers) used as supporting material with sugar-free Murashige and Skoog liquid medium with double-strength KH₂PO₄, MgSO₄, FeSO₄, and Na₂-EDTA in the vessel with enhanced natural ventilation. CO₂ concentration in the culture room was kept at 1500 μmol mol⁻¹ (four times the atmospheric CO₂ concentration) during the photoperiod. A factorial experiment was designed with two levels of photosynthetic photon flux (PPF) and three levels of N (number of air exchanges of the vessel). The results were compared with those in the control treatment (photomixotrophic culture using sugar-containing agar medium under low PPF and low N). PPF and N showed significant positive effects on the growth of sugarcane plantlets in vitro. In the photoautotrophic (using sugar-free medium) treatments with relatively high PPF (200–400 μmol m⁻² s⁻¹) and high N (2–10 h⁻¹), the growth of plantlets was four to seven times greater than that in the control. Also, the culture period for multiplication and rooting was shortened from 30 d in the control to 18 d or less in the photoautotrophic, high PPF, and high N treatments. Use of porous supporting material in photoautotrophic treatments promoted rooting and plantlet growth significantly.     

Chemical constituents isolated from Paeonia lactiflora roots and their neuroprotective activity against oxidative stress in vitro

The methanolic extract of Paeonia lactiflora roots significantly protected primary cultures of rat cortical cells exposed to oxidative stress induced by H₂O₂. Seven monoterpenes, paeonilactone-B (1), paeonilactone-C (2), paeoniflorigenone (3), benzoylpaeoniflorin (4), paeoniflorin (5), oxypaeoniflorin (6) and albiflorin (7), were isolated by bioactivity-guided fractionation and further separation using chromatographic techniques. Among them, compounds 2 and 4 significantly protected primary cultures of rat cortical cells against H₂O₂-induced neurotoxicity.     

Alpha-melanocyte stimulating hormone protects retinal pigment epithelium cells from oxidative stress through activation of melanocortin 1 receptor–Akt–mTOR signaling

Patients with age related macular degeneration (AMD) will develop vision loss in the center of the visual field. Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is an important contributor of AMD. In this study, we explored the pro-survival effect of α-melanocyte stimulating hormone (α-MSH) on oxidative stressed RPE cells. We found that α-MSH receptor melanocortin 1 receptor (MC1R) was functionally expressed in primary and transformed RPE cells. RPE cells were response to α-MSH stimulation. α-MSH activated Akt/mammalian target of rapamycin (mTOR) and Erk1/2 signalings in RPE cells, which were inhibited by MC1R siRNA knockdown. α-MSH protected RPE cells from hydrogen peroxide (H₂O₂)-induced apoptosis, an effect that was almost abolished when MC1R was depleted by siRNA. α-MSH-mediated S6K1 activation and pro-survival effect against H₂O₂ was inhibited by Akt inhibitors (perifosine, MK-2206 and LY294002). Further, mTOR inhibition by rapamycin, or by mTOR siRNA knockdown, diminished α-MSH’s pro-survival effect in RPE cells. Thus, Akt and its downstream mTOR signaling mediates α-MSH-induced survival in RPE cells. In summary, we have identified a new α-MSH–MC1R physiologic pathway that reduces H₂O₂-induced RPE cell damage, and might minimize the risk of developing AMD.     

Interaction of allosteric effectors (ATP,CO2, H+) modulating oxygen affinity of the hemoglobin in the carp,Cyprinus carpio,in vitro

Summary The interaction of allosteric effectors (CO₂, ATP, H⁺) with respect to the oxygen affinity of carp hemoglobin was analyzed by determining oxygen binding curves spectrophotometrically in dilute solutions of stripped hemoglobin at 20°C. The pH range studied was 6.8–8.2.P _CO2 was 0, 10 and 70 mmHg (0, 1.33 and 9.3 kPa). ATP/Hb₄ was 0, 8 and 24. In the presence of either CO₂ or ATP, the effects of the cofactors onP ₅₀ were as expected over the whole pH range. In contrast to other published data, each cofactor also had a significant effect onP ₅₀ in the presence of the other cofactor. Evidence was obtained that oxylabile carbamate is formed by carp hemoglobin and that the formation of carbamate persists at a lower level in the presence of ATP. The results support the view that the binding of ATP to carp hemoglobin requires only one terminal amino group, leaving the other N-terminal of the -chain free to react with CO₂.     

Isolation and Characterization of Antibacterial Compound from a Mangrove-Endophytic Fungus, Penicillium chrysogenum MTCC 5108

Microorganisms, especially endophytic fungi that reside in the tissue of living mangrove plants, seem to play a major role in meeting the general demand for new biologically active substances. During the course of screening for biologically active secondary metabolites from marine microorganisms, an antibiotic compound containing an indole and a diketopiperazine moiety was isolated from the culture medium of Penicilliumchrysogenum, (MTCC 5108), an endophytic fungus on the mangrove plant Porteresiacoarctata (Roxb.). The cell free culture medium of P. chrysogenum showed significant activity against Vibriocholerae, (MCM B-322), a pathogen causing cholera in humans. Bioassay guided chemical characterization of the crude extract led to the isolation of a secondary metabolite possessing a molecular formula C₁₉H₂₁O₂N₃. Its antibacterial activity was comparable with standard antibiotic, streptomycin. This compound (1) was found to be (3,1′-didehydro-3[2″(3′″,3′″-dimethyl-prop-2-enyl)-3″-indolylmethylene]-6-methyl pipera-zine-2,5-dione) on the basis of mass spectrometry, infrared spectroscopy and one and two-dimensional nuclear magnetic resonance analysis.     

Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, potentiates ATP-gated P2X1 receptor channel currents

Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X₁–P2X₇) have been identified. Most of the P2X₁ receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X₁ receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X₁ receptors. In the present study, we examined the effect of gintonin on P2X₁ receptor channel activity. P2X₁ receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X₁ receptors induced large inward currents (I _ATP ), but repetitive ATP treatments induced a rapid desensitization of I _ATP . Gintonin treatment after P2X₁ receptor desensitization potentiated I _ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I _ATP . Gintoninmediated I _ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X₁ receptor greatly attenuated the gintonin-mediated I _ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X₁ receptor through a phosphoinositide-dependent pathway.     

The rate-limiting step of sulfiredoxin is associated with the transfer of the γ-phosphate of ATP to the sulfinic acid of overoxidized typical 2-Cys peroxiredoxins

The eukaryotic sulfiredoxin (Srx) catalyzes the reduction of overoxidized typical 2-Cys peroxiredoxins PrxSO₂ via ATP/Mg²⁺-dependent phosphorylation of the sulfinic acid group, followed by formation of a PrxSO-SSrx thiolsulfinate intermediate. Using real-time kinetics of wild-type and C84A Srxs, and pH-rate profiles with ATP/Mg²⁺ analogues, we show that the rate-limiting step of the reaction is associated with the chemical process of transfer of the γ-phosphate of ATP to the sulfinic acid, in contrast to that described by Jönsson et al. [7]. Two pK_apps of 6.2 and 7.5 were extracted from the bell-shaped pH-rate profile, corresponding to the γ-phosphate of ATP, and to an acid–base catalyst, respectively.