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1.
Inhibition of Chemoautotrophic Nitrification by Sodium Chlorate and Sodium Chlorite: a Reexamination 总被引:3,自引:5,他引:3
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The oxidation of NH4+ by Nitrosomonas europaea was insensitive to 10 mM NaClO3 (sodium chlorate) but was strongly inhibited by NaClO2 (sodium chlorite; Ki, 2 μM). The oxidation of NO2− by Nitrobacter winogradskyi was inhibited by both ClO3− and ClO2− (Ki for ClO2−, 100 μM). N. winogradskyi reduced ClO3− to ClO2− under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO2− was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH4+ and NO2− was inhibited in the presence of 10 mM ClO3− after a 2-h lag period, despite the fact that, under these conditions, ClO2− was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH4+ oxidation is inhibited by ClO2− produced by reduction of ClO3− by the NO2− oxidizer. The use of ClO3− inhibition of NO2− oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH4+ is not affected. 相似文献
2.
Sang Su Kwak Mikyoung Jeong Ji Hye Choi Daesoo Kim Hyesun Min Yoosik Yoon Onyou Hwang Gary G. Meadows Cheol O. Joe 《PloS one》2013,8(4)
This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH4)-deficient Spr
−/− mice by the dietary supplementation of tyrosine. Since BH4 is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH4-deficient Spr
−/− mice. We found that Spr
−/− mice display variable ‘open-field’ behaviors, impaired motor functions on the ‘rotating rod’, and dystonic ‘hind-limb clasping’. In this study, we report that these aberrant motor deficits displayed by Spr
−/− mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of Spr
−/− mice may not be the biological feature of aberrant motor behaviors associated with BH4 deficiency. Brain levels of dopamine (DA) and its metabolites in Spr
−/− mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of Spr
−/− mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH4-deficiency. 相似文献
3.
Nicole Wydeven Ekaterina Posokhova Zhilian Xia Kirill A. Martemyanov Kevin Wickman 《The Journal of biological chemistry》2014,289(4):2440-2449
Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K+ channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4−/−, Rgs6−/−, and Rgs4−/−:Rgs6−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6−/− and Gβ5−/− mice, suggest that the partial rescue of phenotypes in Rgs4−/−:Rgs6−/− mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice. 相似文献
4.
Gaspar Pe?a-Münzenmayer Marcelo A. Catalán Yusuke Kondo Yasna Jaramillo Frances Liu Gary E. Shull James E. Melvin 《The Journal of biological chemistry》2015,290(17):10677-10688
Transcellular Cl− movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na+-K+-2Cl− cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl− above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl− uptake pathway concentrates Cl− ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl−/HCO3− exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4−/− mice. The decrease in salivation was not related to loss of Na+-K+-2Cl− cotransporter or Na+/H+ exchanger activity in Ae4−/− mice but correlated with reduced Cl− uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl−/HCO3− exchanger activity revealed that HCO3−-dependent Cl− uptake was reduced in the acinar cells of Ae2−/− and Ae4−/− mice. Moreover, Cl−/HCO3− exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl−/HCO3− exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion. 相似文献
5.
The δ13C value of a tropical marine grass Thalassia testudinum is −9.04‰. This value is similar to the δ13C value of terrestrial tropical grasses. The δ13C values of the organic acid fraction, the amino acid fraction, the sugar fraction, malic acid, and glucose are: −11.2‰, −13.1‰, −10.1‰, −11.1‰, and −11.5‰, respectively. The δ13C values of malic acid and glucose of Thalassia are similar to the δ13C values of these intermediates in sorghum leaves and attest to the presence of the photosynthetic C4-dicarboxylic acid pathway in this marine grass. The inorganic HCO3− for the growth of the grass fluctuates between −6.7 to −2.7‰ during the day. If CO2 fixation in Thalassia is catalyzed by phosphoenolpyruvate carboxylase (which would result in a −3‰ fractionation between HCO3− and malic acid), the predicted δ13C value for Thalassia would be −9.7 to −5.7‰. This range is close to the observed range of −12.6 to −7.8‰ for Thalassia and agree with the operation of the C4-dicarboxylic acid pathway in this plant. The early products of the fixation of HCO3− in the leaf sections are malic acid and aspartic acid which are similar to the early products of CO2 fixation in C4 terrestrial plants. 相似文献
6.
Studies of human NK cells and their role in tumor suppression have largely been restricted to in vitro experiments which lack the complexity of whole organisms, or mouse models which differ significantly from humans. In this study we showed that, in contrast to C57BL/6 Rag2−/−/γc
−/− and NOD/Scid mice, newborn BALB/c Rag2−/−/γc
−/− mice can support the development of human NK cells and CD56+ T cells after intrahepatic injection with hematopoietic stem cells. The human CD56+ cells in BALB/c Rag2−/−/γc
−/− mice were able to produce IFN-γ in response to human IL-15 and polyI:C. NK cells from reconstituted Rag2−/−/γc
−/− mice were also able to kill and inhibit the growth of K562 cells in vitro and were able to produce IFN-γ in response to stimulation with K562 cells. In vivo, reconstituted Rag2−/−/γc
−/− mice had higher survival rates after K562 challenge compared to non-reconstituted Rag2−/−/γc
−/− mice and were able to control tumor burden in various organs. Reconstituted Rag2−/−/γc
−/− mice represent a model in which functional human NK and CD56+ T cells can develop from stem cells and can thus be used to study human disease in a more clinically relevant environment. 相似文献
7.
Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO3−, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO3−in vivo, −NO3−in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO3− to the in vivo NR assay medium increased activity (over that of the −NO3−in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO3− was rate-limiting. The stimulation of in vivo NR activity by NO3− was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO3− was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO2− [g fresh weight, hr]−1) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting. 相似文献
8.
Volkan Seyrantepe Pablo Lema Aurore Caqueret Larbi Dridi Samar Bel Hadj Stephane Carpentier Francine Boucher Thierry Levade Lionel Carmant Roy A. Gravel Edith Hamel Pascal Vachon Graziella Di Cristo Jacques L. Michaud Carlos R. Morales Alexey V. Pshezhetsky 《PLoS genetics》2010,6(9)
Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts GM2 to GM3 ganglioside. Hexa−/− mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise GM2 ganglioside via a lysosomal sialidase into glycolipid GA2, which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4
−/−;Hexa
−/−) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa
−/− or Neu4
−/− siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating GM2 ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa−/− mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa
−/− mice. 相似文献
9.
Evapotranspiration (E) and CO2 flux (Fc) in the growing season of an unusual dry year were measured continuously over a Scots pine forest in eastern Finland, by eddy covariance techniques. The aims were to gain an understanding of their biological and environmental control processes. As a result, there were obvious diurnal and seasonal changes in E, Fc, surface conductance (gc), and decoupling coefficient (Ω), showing similar trends to those in radiation (PAR) and vapour pressure deficit (δ). The maximum mean daily values (24-h average) for E, Fc, gc, and Ω were 1.78 mmol m−2 s−1, −11.18 µmol m−2 s−1, 6.27 mm s−1, and 0.31, respectively, with seasonal averages of 0.71 mmol m−2 s−1, −4.61 µmol m−2 s−1, 3.3 mm s−1, and 0.16. E and Fc were controlled by combined biological and environmental variables. There was curvilinear dependence of E on gc and Fc on gc. Among the environmental variables, PAR was the most important factor having a positive linear relationship to E and curvilinear relationship to Fc, while vapour pressure deficit was the most important environmental factor affecting gc. Water use efficiency was slightly higher in the dry season, with mean monthly values ranging from 6.67 to 7.48 μmol CO2 (mmol H2O)−1 and a seasonal average of 7.06 μmol CO2 (μmol H2O)−1. Low Ω and its close positive relationship with gc indicate that evapotranspiration was sensitive to surface conductance. Mid summer drought reduced surface conductance and decoupling coefficient, suggesting a more biotic control of evapotranspiration and a physiological acclimation to dry air. Surface conductance remained low and constant under dry condition, supporting that a constant value of surface constant can be used for modelling transpiration under drought condition. 相似文献
10.
Jae Kyu Lim Seung Seob Bae Tae Wan Kim Jung-Hyun Lee Hyun Sook Lee Sung Gyun Kang 《Applied and environmental microbiology》2012,78(20):7393-7397
Formate-dependent proton reduction to H2 (HCOO− + H2O → HCO3− + H2) has been reported for hyperthermophilic Thermococcus strains. In this study, a hyperthermophilic archaeon, Thermococcus onnurineus strain NA1, yielded H2 accumulation to a partial pressure of 1 × 105 to 7 × 105 Pa until the values of Gibbs free energy change (ΔG) reached near thermodynamic equilibrium (−1 to −3 kJ mol−1). The bioenergetic requirement for the metabolism to conserve energy was demonstrated by ΔG values as small as −5 kJ mol−1, which are less than the biological minimum energy quantum, −20 kJ mol−1, as calculated by Schink (B. Schink, Microbiol. Mol. Biol. Rev. 61:262-280, 1997). Considering formate as a possible H2 storage material, the H2 production potential of the strain was assessed. The volumetric H2 production rate increased linearly with increasing cell density, leading to 2,820 mmol liter−1 h−1 at an optical density at 600 nm (OD600) of 18.6, and resulted in the high specific H2 production rates of 404 ± 6 mmol g−1 h−1. The H2 productivity indicates the great potential of T. onnurineus strain NA1 for practical application in comparison with H2-producing microbes. Our result demonstrates that T. onnurineus strain NA1 has a highly efficient metabolic system to thrive on formate in hydrothermal systems. 相似文献
11.
The reversibility of adenosine triphosphate cleavage by myosin 总被引:12,自引:12,他引:0
For the simplest kinetic model the reverse rate constants (k−1 and k−2) associated with ATP binding and cleavage on purified heavy meromyosin and heavy meromyosin subfragment 1 from rabbit skeletal muscle in the presence of 5mm-MgCl2, 50mm-KCl and 20mm-Tris–HCl buffer at pH8.0 and 22°C are: k−1<0.02s−1 and k−1=16s−1. Apparently, higher values of k−1 and k−2 are found with less-purified protein preparations. The values of k−1 and k−2 satisfy conditions required by previous 18O-incorporation studies of H218O into the Pi moiety on ATP hydrolysis and suggest that the cleavage step does involve hydrolysis of ATP or formation of an adduct between ATP and water. The equilibrium constant for the cleavage step at the myosin active site is 9. If the cycle of events during muscle contraction is described by the model proposed by Lymn & Taylor (1971), the fact that there is only a small negative standard free-energy change for the cleavage step is advantageous for efficient chemical to mechanical energy exchange during muscle contraction. 相似文献
12.
A modification of the Adler capillary assay was used to evaluate the chemotactic responses of several denitrifiers to nitrate and nitrite. Strong positive chemotaxis was observed to NO3− and NO2− by soil isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri, with the peak response occurring at 10−3 M for both attractants. In addition, a strong chemoattraction to serine (peak response at 10−2 M), tryptone, and a soil extract, but not to NH4+, was observed for all denitrifiers tested. Chemotaxis was not dependent on a previous growth on NO3−, NO2−, or a soil extract, and the chemoattraction to NO3− occurred when the bacteria were grown aerobically or anaerobically. However, the best response to NO3− was usually observed when the cells were grown aerobically with 10 mM NO3− in the growth medium. Capillary tubes containing 10−3 M NO3− submerged into soil-water mixtures elicited a significant chemotactic response to NO3− by the indigenous soil microflora, the majority of which were Pseudomonas spp. A chemotactic strain of P. fluorescens also was shown to survive significantly better in aerobic and anaerobic soils than was a nonmotile strain of the same species. Both strains had equal growth rates in liquid cultures. Thus, chemotaxis may be one mechanism by which denitrifiers successfully compete for available NO3− and NO2−, and which may facilitate the survival of naturally occurring populations of some denitrifiers. 相似文献
13.
Soline Bourgeois Lisa Bounoure Erik I. Christensen Suresh K. Ramakrishnan Pascal Houillier Olivier Devuyst Carsten A. Wagner 《The Journal of biological chemistry》2013,288(8):5518-5529
Ammonia secretion by the collecting duct (CD) is critical for acid-base homeostasis and, when defective, causes distal renal tubular acidosis (dRTA). The Rhesus protein RhCG mediates NH3 transport as evident from cell-free and cellular models as well as from Rhcg-null mice. Here, we investigated in a Rhcg mouse model the metabolic effects of Rhcg haploinsufficiency, the role of Rhcg in basolateral NH3 transport, and the mechanisms of adaptation to the lack of Rhcg. Both Rhcg+/+ and Rhcg+/− mice were able to handle an acute acid load, whereas Rhcg−/− mice developed severe metabolic acidosis with reduced ammonuria and high mortality. However, chronic acid loading revealed that Rhcg+/− mice did not fully recover, showing lower blood HCO3− concentration and more alkaline urine. Microperfusion studies demonstrated that transepithelial NH3 permeability was reduced by 80 and 40%, respectively, in CDs from Rhcg−/− and Rhcg+/− mice compared with controls. Basolateral membrane permeability to NH3 was reduced in CDs from Rhcg−/− mice consistent with basolateral Rhcg localization. Rhcg−/− responded to acid loading with normal expression of enzymes and transporters involved in proximal tubular ammoniagenesis but reduced abundance of the NKCC2 transporter responsible for medullary accumulation of ammonium. Consequently, tissue ammonium content was decreased. These data demonstrate a role for apical and basolateral Rhcg in transepithelial NH3 transport and uncover an incomplete dRTA phenotype in Rhcg+/− mice. Haploinsufficiency or reduced expression of RhCG may underlie human forms of (in)complete dRTA. 相似文献
14.
Transformation of Industrial Dyes by Manganese Peroxidases from Bjerkandera adusta and Pleurotus eryngii in a Manganese-Independent Reaction 总被引:6,自引:0,他引:6
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A. Heinfling M. J. Martínez A. T. Martínez M. Bergbauer U. Szewzyk 《Applied microbiology》1998,64(8):2788-2793
We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn3+-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn2+. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn2+-independent manner. The reactions with the dyes were characterized by apparent Km values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn2+, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn3+-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1. 相似文献
15.
Comparative Kinetics and Reciprocal Inhibition of Nitrate and Nitrite Uptake in Roots of Uninduced and Induced Barley (Hordeum vulgare L.) Seedlings 总被引:9,自引:5,他引:4
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Nitrate and NO2− transport by roots of 8-day-old uninduced and induced intact barley (Hordeum vulgare L. var CM 72) seedlings were compared to kinetic patterns, reciprocal inhibition of the transport systems, and the effect of the inhibitor, p-hydroxymercuribenzoate. Net uptake of NO3− and NO2− was measured by following the depletion of the ions from the uptake solutions. The roots of uninduced seedlings possessed a low concentration, saturable, low Km, possibly a constitutive uptake system, and a linear system for both NO3− and NO2−. The low Km system followed Michaelis-Menten kinetics and approached saturation between 40 and 100 micromolar, whereas the linear system was detected between 100 and 500 micromolar. In roots of induced seedlings, rates for both NO3− and NO2− uptake followed Michaelis-Menten kinetics and approached saturation at about 200 micromolar. In induced roots, two kinetically identifiable transport systems were resolved for each anion. At the lower substrate concentrations, less than 10 micromolar, the apparent low Kms of NO3− and NO2− uptake were 7 and 9 micromolar, respectively, and were similar to those of the low Km system in uninduced roots. At substrate concentrations between 10 and 200 micromolar, the apparent high Km values of NO3− uptake ranged from 34 to 36 micromolar and of NO2− uptake ranged from 41 to 49 micromolar. A linear system was also found in induced seedlings at concentrations above 500 micromolar. Double reciprocal plots indicated that NO3− and NO2− inhibited the uptake of each other competitively in both uninduced and induced seedlings; however, Ki values showed that NO3− was a more effective inhibitor than NO2−. Nitrate and NO2− transport by both the low and high Km systems were greatly inhibited by p-hydroxymercuribenzoate, whereas the linear system was only slightly inhibited. 相似文献
16.
Kristen R. Vella Preeti Ramadoss Ricardo H. Costa-e-Sousa Inna Astapova Felix D. Ye Kaila A. Holtz Jamie C. Harris Anthony N. Hollenberg 《Molecular and cellular biology》2014,34(9):1564-1575
Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoRΔID/ΔID
Src-1−/− mice have normal TH and TSH levels and are triiodothryonine (T3) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T3 activation of key hepatic gene targets, NCoRΔID/ΔID
Src-1−/− mice reacquired hepatic T3 sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoRΔID/ΔID
Src-1−/− mice, suggesting that SRC-2 is responsible for T3 sensitivity in the absence of NCoR1 and SRC-1. Thus, T3 targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1−/− mice through increased SRC-2 recruitment to T3 target genes. 相似文献
17.
Justin M. Bradley Sophie J. Marritt Margaret A. Kihlken Kate Haynes Andrew M. Hemmings Ben C. Berks Myles R. Cheesman Julea N. Butt 《The Journal of biological chemistry》2012,287(48):40350-40359
SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (Em) at pH 7.0 of approximately +210, −340, and −400 mV for the His/Met, His/Cys−, and active site His/CysS−-ligated heme, respectively. Exposing SoxAX to S2O42−, a substrate analog with Em ∼−450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (Em ∼−1140 mV), allows cyanide to displace the cysteine persulfide (CysS−) ligand to the active site heme. This provides the first evidence for the dissociation of CysS− that has been proposed as a key event in SoxAX catalysis. 相似文献
18.
Winifred O. Idigo Svetlana Reilly Mei Hua Zhang Yin Hua Zhang Raja Jayaram Ricardo Carnicer Mark J. Crabtree Jean-Luc Balligand Barbara Casadei 《The Journal of biological chemistry》2012,287(52):43665-43673
Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β3-adrenergic receptor (β3-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β3-AR signaling and found that the β3-AR-mediated reduction in cell shortening and [Ca2+]i transient amplitude was abolished both in eNOS−/− and nNOS−/− left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-l-thiocitrulline. LV superoxide (O2˙̄) production was increased in nNOS−/− mice and reduced by l-Nω-nitroarginine methyl ester (l-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS−/− myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O2˙̄, only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca2+]i transient response to β3-AR stimulation in nNOS−/− mice. In summary, our data show that increased O2˙̄ production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β3-AR stimulation in nNOS−/− myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS−/− mice result from disruption of eNOS signaling. 相似文献
19.
Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate 总被引:2,自引:1,他引:1
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The H+-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl− and NO3−. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the Km of the enzyme for ATP but caused relatively little alteration of the Vmax. Nitrate increased Km only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl−. Other monovalent anions that specifically increased the Km of the ATPase for ATP were, in order of increasing Ki, SCN−, ClO4−, and ClO3−. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO3−, was a competitive inhibitor of the ATPase but ADP and NO3− did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO3− does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO3− exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl− nor NO3− are involved in substrate binding. 相似文献