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1.
J Bacteriol ; 201(16)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31160396

RESUMO

Runella slithyformis HD-Pnk is the prototype of a family of dual 5' and 3' nucleic acid end-healing enzymes that phosphorylate 5'-OH termini and dephosphorylate 2',3'-cyclic-PO4, 3'-PO4, and 2'-PO4 ends. HD-Pnk is composed of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Here, we probed the phosphoesterase activity of HD-Pnk by querying its ability to hydrolyze non-nucleic acid phosphoester substrates and by conducting a mutational analysis of conserved amino acid constituents of the HD domain. We report that HD-Pnk catalyzes vigorous hydrolysis of p-nitrophenylphosphate (Km = 3.13 mM; k cat = 27.8 s-1) using copper as its metal cofactor. Mutagenesis identified Gln28, His33, His73, Asp74, Lys77, His94, His127, Asp162, and Arg166 as essential for p-nitrophenylphosphatase and DNA 3' phosphatase activities. Structural modeling places these residues at the active site, wherein His33, His73, Asp74, His94, and His127 are predicted to coordinate a binuclear metal complex and Lys77 and Arg166 engage the scissile phosphate. HD-Pnk homologs are distributed broadly (and exclusively) in bacteria, usually in a two-gene cluster with a putative ATP-dependent polynucleotide ligase (LIG). We speculate that HD-Pnk and LIG comprise the end-healing and end-sealing components of a bacterial nucleic acid repair pathway.IMPORTANCE 5'-end healing and 3'-end healing are key steps in nucleic acid break repair in which 5'-OH ends are phosphorylated by a polynucleotide kinase, and 3'-PO4 or 2',3'-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate 5'-PO4 and 3'-OH termini needed for joining by DNA and RNA ligases. This study interrogates, biochemically and via mutagenesis, the phosphoesterase activity of Runella slithyformis HD-Pnk, a bifunctional bacterial 5'- and 3'-end-healing enzyme composed of HD phosphoesterase and P-loop kinase modules. HD-Pnk homologs are found in 129 bacterial genera from 11 phyla. In 123/129 instances, HD-Pnk is encoded in an operon-like gene cluster with a putative ATP-dependent polynucleotide ligase (LIG), suggesting that HD-Pnk and LIG are agents of a conserved bacterial nucleic acid repair pathway.


Assuntos
4-Nitrofenilfosfatase/química , 4-Nitrofenilfosfatase/metabolismo , Proteínas de Bactérias/química , Cytophagaceae/enzimologia , Polinucleotídeo 5'-Hidroxiquinase/química , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , 4-Nitrofenilfosfatase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cobre/metabolismo , Cytophagaceae/química , Cytophagaceae/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Óperon , Polinucleotídeo 5'-Hidroxiquinase/genética , Domínios Proteicos , Alinhamento de Sequência
2.
ACS Appl Mater Interfaces ; 11(1): 195-201, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30556997

RESUMO

Nanoceria with phosphatase-like behavior shows its great potential for many important biological applications through a catalytic dephosphorylation process. Herein, we synthesize a series of porous nanorods of ceria (PN-CeO2) with the controllable surface Ce3+ fractions modulated by thermal annealing, understanding the correlations between their surface properties and reactivity for the dephosphorylation of p-nitrophenyl phosphate ( p-NPP) and investigating their catalytic performance under various interferences. Our results suggest that PN-CeO2 with abundant surface defects deliver higher catalytic activity to break down p-NPP. Most importantly, PN-CeO2 exhibited a better adaptability over a wide pH range and preserved the catalytic activity over a wide temperature range from 20 to 80 °C, if compared with natural enzymes. Moreover, PN-CeO2 delivered the high catalytic stability against various interference ions. Their great prospects for practical applications were further demonstrated by dephosphorylation of DNA.


Assuntos
4-Nitrofenilfosfatase/química , Cério/química , Temperatura Alta , Nanotubos/química , Nitrofenóis/química , Compostos Organofosforados/química , Catálise , Concentração de Íons de Hidrogênio , Porosidade
3.
J Biosci Bioeng ; 119(3): 297-302, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25282639

RESUMO

The hydrolysis of lignocellulosic biomass liberates sugars, primarily glucose and xylose, which are subsequently converted to ethanol by microbial fermentation. The rapid and efficient fermentation of xylose by recombinant Saccharomyces cerevisiae strains is limited by weak acids generated during biomass pretreatment processes. In particular, acetic acid negatively affects cell growth, xylose fermentation rate, and ethanol production. The ability of S. cerevisiae to efficiently utilize xylose in the presence of acetic acid is an essential requirement for the cost-effective production of ethanol from lignocellulosic hydrolysates. Here, an acetic acid-responsive transcriptional activator, HAA1, was overexpressed in a recombinant xylose-fermenting S. cerevisiae strain to yield BY4741X/HAA1. This strain exhibited improved cell growth and ethanol production from xylose under aerobic and oxygen limited conditions, respectively, in the presence of acetic acid. The HAA1p regulon enhanced transcript levels in BY4741X/HAA1. The disruption of PHO13, a p-nitrophenylphosphatase gene, in BY4741X/HAA1 led to further improvement in both yeast growth and the ability to ferment xylose, indicating that HAA1 overexpression and PHO13 deletion act by different mechanisms to enhance ethanol production.


Assuntos
Ácido Acético/metabolismo , Etanol/metabolismo , Fermentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , 4-Nitrofenilfosfatase/genética , Ácido Acético/farmacologia , Aerobiose , Meios de Cultura/química , Fermentação/efeitos dos fármacos , Expressão Gênica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
4.
Biotechnol Lett ; 36(10): 2011-21, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24966040

RESUMO

In the industrial production of bioethanol from lignocellulosic biomass, a strain of Saccharomyces cerevisiae that can ferment xylose in the presence of inhibitors is of utmost importance. The recombinant, industrial-flocculating S. cerevisiae strain NAPX37, which can ferment xylose, was used as the parent to delete the gene encoding p-nitrophenylphosphatase (PHO13) and overexpress the gene encoding transaldolase (TAL1) to evaluate the synergistic effects of these two genes on xylose fermentation in the presence of weak acid inhibitors, including formic, acetic, or levulinic acids. TAL1 over-expression or PHO13 deletion improved xylose fermentation as well as the tolerance of NAPX37 to all three weak acids. The simultaneous deletion of PHO13 and the over-expression of TAL1 had synergistic effects and improved ethanol production and reduction of xylitol accumulation in the absence and presence of weak acid inhibitors.


Assuntos
Microbiologia Industrial , Saccharomyces cerevisiae/genética , Xilose/metabolismo , 4-Nitrofenilfosfatase/genética , Ácido Acético/metabolismo , Fermentação , Formiatos/metabolismo , Deleção de Genes , Expressão Gênica , Ácidos Levulínicos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Transaldolase/genética
5.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 6): 697-702, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24915075

RESUMO

Using directed evolution based on random mutagenesis and heat-treated selection, a thermostable His170Tyr mutant of Geobacillus stearothermophilus thermostable p-nitrophenylphosphatase (TpNPPase) was obtained. The temperature at which the His170Tyr mutant lost 50% of its activity (T1/2) was found to be 4.40 K higher than that of wild-type TpNPPase, and the melting temperature of the His170Tyr mutant increased by 2.39 K. The crystal structure of the His170Tyr mutant was then determined at 2.0 Šresolution in the presence of a sodium ion and a sulfate ion in the active site. The cap domain of chain B shows a half-closed conformation. The hydrophobic side chain of the mutated residue, the hydroxyphenyl group, forms a hydrophobic contact with the methyl group of Ala166. This hydrophobic interaction was found using the Protein Interactions Calculator (PIC) web server with an interaction distance of 4.6 Å, and might be a key factor in the thermostabilization of the His170Tyr mutant. This study potentially offers a molecular basis for both investigation of the catalytic mechanism and thermostable protein engineering.


Assuntos
4-Nitrofenilfosfatase/metabolismo , Geobacillus stearothermophilus/enzimologia , Histidina/química , Tirosina/química , Mutação
6.
PLoS One ; 9(5): e96909, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24816799

RESUMO

Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.


Assuntos
Capsaicina/análogos & derivados , Domínio Catalítico , Membrana Celular/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , 4-Nitrofenilfosfatase/antagonistas & inibidores , 4-Nitrofenilfosfatase/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Capsaicina/farmacologia , Membrana Celular/enzimologia , Endopeptidase K/metabolismo , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Isoxazóis/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteólise/efeitos dos fármacos , Ratos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , Temperatura , Tripsina/metabolismo , Vanadatos/farmacologia
7.
Protein Pept Lett ; 21(5): 483-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24256105

RESUMO

Thermostable p-nitrophenylphosphatase from Bacillus Stearothermophilus (Bs-TpNPPase) is involved in the Mg(2+)-dependent hydrolysis of the phosphoenzyme at an optimum reaction temperature of 55°C. Bs-TpNPPase has been cloned and overexpressed in the E.coli M15 strain. Based on the conserved active sites, the protein was suggested to be a member of the haloalkanoate dehalogenase (HAD) superfamily. Two site-specific point mutants of Bs-TpNPPase were prepared by changing the catalytic Asp10 and Thr43 to Ala10 and Ala43, respectively. The activity of the two mutants further confirms Bs-TpNPPase as a member of the HAD superfamily. HAD superfamily can be divided into the four subfamilies and play several biochemical roles such as DNA repair, signal transduction and secondary metabolism. To understand the relationship between structure and thermostability in HAD superfamily, Bs-TpNPPase from Bacillus Stearothermophilus was selected. The X-ray crystal structure of Bs-TpNPPase was determined at 1.5A resolution using the molecular replacement phasing method. The structure of Bs-TpNPPase has been deposited and the PDB code is 4KN8. Compared with Bsp, a mesophilic prokaryotic putative p-nitrophenyl phosphatase from Bacillus Subtilis, Bs- TpNPPase showed highly homology but variations in the level of leucine content, aromatic clusters, cation-Pi and hydrophobic interaction. These differences may affect the thermal stability of the protein. The crystal structure of Bs-TpNPPase described herein may serve as a guide to better understand the mechanism of thermostability and provide insights for further mutation work.


Assuntos
4-Nitrofenilfosfatase/química , Geobacillus stearothermophilus/enzimologia , 4-Nitrofenilfosfatase/genética , Cristalografia por Raios X , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estabilidade Proteica , Temperatura
8.
J Biosci Bioeng ; 116(6): 706-15, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23810666

RESUMO

A recombinant xylose-utilizing Saccharomyces cerevisiae strain carrying one copy of heterologous XYL1 and XYL2 from Pichia stipitis and endogenous XKS1 under the control of the TDH3 promoter in the chromosomal DNA was constructed from the industrial haploid yeast strain NAM34-4C, which showed thermotolerance and acid tolerance. The recombinant S. cerevisiae strain SCB7 grew in minimal medium containing xylose as the sole carbon source, and its shortest generation time (G(short)) was 5 h. From this strain, four mutants showing rapid growth (G(short) = 2.5 h) in the minimal medium were isolated. The mutants carried four mutations that were classified into three linkage groups. Three mutations were dominant and one mutation was recessive to the wild type allele. The recessive mutation was in the PHO13 gene encoding para-nitrophenyl phosphatase. The other mutant genes were not linked to TAL1 gene encoding transaldolase. When the mutants and their parental strain were used for the batch fermentation in a complex medium at pH 4.0 containing 30 g/L xylose at 35 °C with shaking (60 rpm) and an initial cell density (Absorbance at 660 nm) of 1.0, all mutants showed efficient ethanol production and xylose consumption from the early stage of the fermentation culture. In two mutants, within 24 h, 4.8 g/L ethanol was produced, and the ethanol yield was 47%, which was 1.4 times higher than that achieved with the parental strain. The xylose concentration in the medium containing the mutant decreased linearly at a rate of 1 g/L/h until 24 h.


Assuntos
Biocombustíveis/microbiologia , Etanol/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Xilose/metabolismo , 4-Nitrofenilfosfatase/genética , 4-Nitrofenilfosfatase/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Fermentação , Genes Dominantes , Genes Recessivos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaldolase/genética , Transaldolase/metabolismo
9.
J Pain ; 13(9): 836-48, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22901764

RESUMO

UNLABELLED: Peripheral nerve injury generally results in spinal neuronal and glial plastic changes associated with chronic behavioral hypersensitivity. Spinal mitogen-activated protein kinases (MAPKs), eg, p38 or extracellular signal-regulated kinases (ERKs), are instrumental in the development of chronic allodynia in rodents, and new p38 inhibitors have shown potential in acute and neuropathic pain patients. We have previously shown that the cannabinoid type 2 receptor agonist JWH015 inhibits ERK activity by inducing MAPK phosphatase (MKP)-1 and MKP-3 (the major regulators of MAPKs) in vitro in microglial cells. Therefore, we decided to investigate the role of these phosphatases in the mechanisms of action of JWH015 in vivo using the rat L5 nerve transection model of neuropathic pain. We observed that peripheral nerve injury reduced spinal MKP-1/3 expression and activity and that intrathecal JWH015 reduced established L5 nerve injury-induced allodynia, enhanced spinal MKP-1/3 expression and activity, and reduced the phosphorylated form of p38 and ERK-1/2. Triptolide, a pharmacological blocker of MKP-1 and MKP-3 expression, inhibited JWH015's effects, suggesting that JWH015 exerts its antinociceptive effects by modulating MKP-1 and MKP-3. JWH015-induced antinociception and MKP-1 and MKP-3 expression were inhibited by the cannabinoid type 2 receptor antagonist AM630. Our data suggest that MKP-1 and MKP-3 are potential targets for novel analgesic drugs. PERSPECTIVE: MAPKs are pivotal in the development of chronic allodynia in rodent models of neuropathic pain. A cannabinoid type 2 receptor agonist, JWH015, reduced neuropathic allodynia in rats by reducing MAPK phosphorylation and inducing spinal MAPK phosphatases 1 and 3, the major regulators of MAPKs.


Assuntos
Hiperalgesia , Indóis/uso terapêutico , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Neuralgia/complicações , Receptor CB2 de Canabinoide/agonistas , 4-Nitrofenilfosfatase/metabolismo , Animais , Modelos Animais de Doenças , Diterpenos/uso terapêutico , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatase 6 de Especificidade Dupla/metabolismo , Compostos de Epóxi/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Hiperalgesia/patologia , Imunossupressores/uso terapêutico , Indóis/farmacologia , Masculino , Fosfatases da Proteína Quinase Ativada por Mitógeno/classificação , Proteínas do Tecido Nervoso/metabolismo , Fenantrenos/uso terapêutico , Ratos , Ratos Sprague-Dawley , Receptor CB2 de Canabinoide/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Fatores de Tempo
10.
J Negat Results Biomed ; 11: 12, 2012 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-22574873

RESUMO

BACKGROUND: The overstimulation of excitatory glutamatergic neurotransmission and the inhibition of Na(+),K(+)-ATPase enzymatic activity have both been implicated in neurotoxicity and are possibly related to the pathogenesis of epilepsy and neurodegenerative disorders. In the present study, we investigated whether glutamatergic stimulation by the glutamatergic agonists glutamate, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) modulates the Na(+),K(+)-ATPase and the K(+)-p-nitrophenylphosphatase activities in the crude synaptosomal fraction of the hippocampus and the frontal cortex of rats. RESULTS: Our results demonstrated that these glutamatergic agonists did not influence the activities of Na(+),K(+)-ATPase or K(+)-p-nitrophenylphosphatase in the brain structures analyzed. Assays with lower concentrations of ATP to analyze the preferential activity of the Na(+),K(+)-ATPase isoform with high affinity for ATP did not show any influence either. CONCLUSIONS: These findings suggest that under our experimental conditions, the stimulation of glutamatergic receptors does not influence the kinetics of the Na(+),K(+)-ATPase enzyme in the hippocampus and frontal cortex.


Assuntos
4-Nitrofenilfosfatase/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Lobo Frontal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Lobo Frontal/enzimologia , Hipocampo/enzimologia , Ácido Caínico/farmacologia , Masculino , N-Metilaspartato/farmacologia , Ratos , Ratos Wistar , Transmissão Sináptica , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
11.
Proteins ; 79(7): 2146-60, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21538547

RESUMO

The crystal structures of an unliganded and adenosine 5'-monophosphate (AMP) bound, metal-dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis are reported at 2.4 and 1.94 Å, respectively. Functional characterization of this enzyme was guided by computational analysis and then confirmed by experiment. The structure consists of a polymerase and histidinol phosphatase (PHP, Pfam: PF02811) domain with a second domain (residues 105-178) inserted in the middle of the PHP sequence. The insert domain functions in binding AMP, but the precise function and substrate specificity of this domain are unknown. Initial bioinformatics analyses yielded multiple potential functional leads, with most of them suggesting DNA polymerase or DNA replication activity. Phylogenetic analysis indicated a potential DNA polymerase function that was somewhat supported by global structural comparisons identifying the closest structural match to the alpha subunit of DNA polymerase III. However, several other functional predictions, including phosphoesterase, could not be excluded. Theoretical microscopic anomalous titration curve shapes, a computational method for the prediction of active sites from protein 3D structures, identified potential reactive residues in YP_910028.1. Further analysis of the predicted active site and local comparison with its closest structure matches strongly suggested phosphoesterase activity, which was confirmed experimentally. Primer extension assays on both normal and mismatched DNA show neither extension nor degradation and provide evidence that YP_910028.1 has neither DNA polymerase activity nor DNA-proofreading activity. These results suggest that many of the sequence neighbors previously annotated as having DNA polymerase activity may actually be misannotated.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bifidobacterium/enzimologia , Esterases/química , Esterases/metabolismo , 4-Nitrofenilfosfatase/química , 4-Nitrofenilfosfatase/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Cristalografia , DNA Polimerase III/química , DNA Polimerase III/metabolismo , Histidinol-Fosfatase/química , Histidinol-Fosfatase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
12.
Postepy Biochem ; 57(3): 294-303, 2011.
Artigo em Polonês | MEDLINE | ID: mdl-22235655

RESUMO

Purinergic signaling plays an important role in the regulation of many physiological processes. The concentration of nucleotides in extracellular space is controlled by at least two families of nucleotidases: NPPases and NTPDases. These families are examples of convergent evolution of proteins. Above ezymes are not phylogenetically related, but they catalyze the same type of reaction. They hydrolyzed tri- and diphosphonucleosides to monophosphonucleosides and orthophosphate or pyrophosphate. This degradation terminates the nucleotide signaling process and also produces other signaling molecules like ADP, and with 5'-nucleotidase, adenosine. Most of known animal NPPases and NTPDases were found as membranous ectoenzymes or soluble proteins localized in tissue fluids. The aim of this work is to provide information about localization, structure, properties and function of NPPases and NTPDases in the regulation of extracellular concentration of nucleotides and purinergic signaling.


Assuntos
4-Nitrofenilfosfatase/química , 4-Nitrofenilfosfatase/metabolismo , Nucleotídeos/metabolismo , Pirofosfatases/química , Pirofosfatases/metabolismo , Receptores Purinérgicos/metabolismo , Animais , Comunicação Celular/fisiologia , Espaço Extracelular/metabolismo , Humanos
13.
Life Sci ; 86(13-14): 473-81, 2010 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-20149805

RESUMO

AIMS: We sought to identify, purify and partially characterize a protein inhibitor of Na(+)/K(+)-ATPase in cytosol of pulmonary artery smooth muscle. MAIN METHODS: (i) By spectrophotometric assay, we identified an inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na(+)/K(+)-ATPase alpha(2)beta(1) and alpha(1)beta(1) isozymes for determining some characteristics of the inhibitor. KEY FINDINGS: We identified a novel endogenous protein inhibitor of Na(+)/K(+)-ATPase having an apparent mol mass of approximately 70kDa in the cytosolic fraction of the smooth muscle. The IC(50) value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the alpha(2)beta(1) and alpha(1)beta(1) isozymes of the Na(+)/K(+)-ATPase; (ii) it interacted reversibly to the E(1) site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na(+)/K(+)-ATPase exists as (alphabeta)(2) diprotomer. SIGNIFICANCE: The inhibitor binds to the Na(+)/K(+)-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where alpha(2) is more sensitive than alpha(1).


Assuntos
Citosol/metabolismo , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Ouabaína/isolamento & purificação , Ouabaína/metabolismo , 4-Nitrofenilfosfatase/metabolismo , Animais , Bovinos , Citosol/enzimologia , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Músculo Liso/citologia , Músculo Liso/enzimologia , Músculo Liso/metabolismo , Isoformas de Proteínas , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologia , Artéria Pulmonar/metabolismo , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/metabolismo
14.
Physiol Res ; 58(1): 121-126, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18198990

RESUMO

K(+)-p-nitrophenylphosphatase (K(+)pNPPase) is the enzyme, which is considered to be involved in K(+)-dependent hydrolysis of the phosphoenzyme in the reaction cycle of Na(+), K(+)ATPase. The aim of our present study was to characterize some features of K(+)pNPPase in homogenates of the rat brain and liver. We determined p-nitrophenylphosphatase (pNPPase) activity in the presence of various ion combinations (Mg(2+)+ K(+), Mg(2+), K(+)). We found a higher total pNPPase activity in the brain (0.8+/-0.079 nkat/mg protein) than in the liver (0.08+/-0.01 nkat/mg protein). Contrary to the liver, the main part of the total brain activity was K(+)-dependent. The activity of K(+)pNPPase was significantly higher in cerebral cortex homogenates (0.86+/-0.073 nkat/mg protein) in comparison to those of the whole brain (0.57+/-0.075 nkat/mg protein). The specific K(+)pNPPase activity was two times higher in the isolated pellet fraction (0.911+/-0.07 nkat/mg protein), rich in synaptosomes, compared to the whole brain homogenate (0.57+/-0.075 nkat/mg protein). Our results demonstrate the high activity of K(+)pNPPase in the brain tissue and its distribution mainly into the pellet fraction, what might indicate a possible role of K(+)pNPPase in specific structures of the brain, e.g. in synaptosomes.


Assuntos
4-Nitrofenilfosfatase/metabolismo , Encéfalo/enzimologia , Fígado/enzimologia , Animais , Cálcio/metabolismo , Córtex Cerebral/metabolismo , Magnésio/metabolismo , Masculino , Potássio/metabolismo , Ratos , Ratos Wistar , Sinaptossomos/enzimologia
15.
Metab Eng ; 10(6): 360-9, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18249574

RESUMO

Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled bioreactors using engineered S. cerevisiae CEN.PK. Growth on glucose was not significantly affected in pho13Delta mutants, but acetate production increased by 75%. Cell growth, ethanol production, and xylose consumption all increased markedly in pho13Delta mutants. The specific growth rate and rate of specific xylose uptake were approximately 1.5 times higher in the deletion strain than in the parental strain when growing on glucose-xylose mixtures and up to 10-fold higher when growing on xylose alone. In addition to showing higher acetate levels, pho13Delta mutants also produced less glycerol on xylose, suggesting that deletion of Pho13p could improve growth by altering redox levels when cells are grown on xylose.


Assuntos
4-Nitrofenilfosfatase/genética , Etanol/metabolismo , Deleção de Genes , Melhoramento Genético/métodos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/fisiologia , Xilose/metabolismo , 4-Nitrofenilfosfatase/metabolismo , Proliferação de Células , Saccharomyces cerevisiae/citologia
16.
Exp Parasitol ; 119(1): 135-43, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18295760

RESUMO

In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress.


Assuntos
Adenosina Trifosfatases/metabolismo , Temperatura Alta , Leishmania mexicana/enzimologia , 4-Nitrofenilfosfatase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Brasil , Cricetinae , Humanos , Hidrólise , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/parasitologia , Fatores de Tempo , Virulência
17.
Mol Biochem Parasitol ; 157(2): 241-3, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18067979

RESUMO

Thiamine pyrophosphate (TPP), the active form of vitamin B1, is an essential cofactor for several enzymes. Humans depend exclusively on the uptake of vitamin B1, whereas bacteria, plants, fungi and the malaria parasite Plasmodium falciparum are able to synthesise thiamine monophosphate (TMP) de novo. TMP has to be dephosphorylated prior to pyrophosphorylation in order to obtain TPP. In P. falciparum the phosphatase capable to catalyse this reaction has been identified by analysis of the substrate specificity. The recombinant enzyme accepts beside vitamin B1 also nucleotides, phosphorylated sugars and the B6 vitamer pyridoxal 5'-phosphate. Vitamin B1 biosynthesis is known to occur in the cytosol. The cytosolic localisation of this phosphatase was verified by transfection of a GFP chimera construct. Stage specific Northern blot analysis of the phosphatase clearly identified an expression profile throughout the entire erythrocytic life cycle of P. falciparum and thereby emphasises the importance of dephosphorylation reactions within the malaria parasite.


Assuntos
4-Nitrofenilfosfatase/genética , 4-Nitrofenilfosfatase/metabolismo , Plasmodium falciparum/enzimologia , Tiamina Monofosfato/metabolismo , Animais , Citosol/química , DNA de Protozoário/química , DNA de Protozoário/genética , Perfilação da Expressão Gênica , Microscopia de Fluorescência , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Piridoxal/análogos & derivados , Piridoxal/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato , Tiamina/metabolismo
18.
Assay Drug Dev Technol ; 5(5): 645-53, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17939754

RESUMO

Protein phosphatase type 5 (PP5) belongs to the PPP family of serine/threonine protein phosphatases and is expressed in most, if not all, human tissues. Although the physiological roles played by PP5 are not yet clear, PP5 is found in association with several proteins that influence intracellular signaling networks initiated by hormones (i.e., glucocorticoids) or cellular stress (i.e., hypoxia, oxidative stress). Recently, studies conducted with short interfering RNA and antisense oligonucleotides indicate that PP5 plays an important role in the regulation of stress-induced signaling cascades that influence both cell growth and the onset of apoptosis. Therefore, the identification of small molecule inhibitors of PP5 is desired for use in studies to further define the biological/pathological roles of PP5. Such inhibitors may also prove useful for development into novel antitumor agents. Here we describe methods to express and purify large amounts of biologically active PP5c, an inhibitor titration-based assay to determine the amount of PP5 in solution, and a fluorescent phosphatase assay that can be used to screen chemical libraries and natural extracts for the presence of catalytic inhibitors.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/biossíntese , 4-Nitrofenilfosfatase/metabolismo , Catálise , Inibidores Enzimáticos/química , Escherichia coli/metabolismo , Corantes Fluorescentes , Humanos , Himecromona/análogos & derivados , Himecromona/química , Indicadores e Reagentes , Proteínas Nucleares/isolamento & purificação , Fosfoproteínas Fosfatases/isolamento & purificação , Espectrometria de Fluorescência
19.
J Membr Biol ; 216(1): 49-59, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17572836

RESUMO

The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr(2+) and Ba(2+) did not compete with K(+) for ATPase (an extracellular K(+) effect). Sr(2+) and Ba(2+) did compete with Na(+) for ATPase (an intracellular Na(+) effect) and with K(+) for pNPPase (an intracellular K(+) effect). These results suggest that Ba(2+) or Sr(2+) can bind to the intracellular transport site, yet neither Ba(2+) nor Sr(2+) was able to activate pNPPase activity; we confirmed that Ca(2+) and Mn(2+) did activate. As another measure of cation binding, we observed that Ca(2+) and Mn(2+), but not Ba(2+), decreased the fluorescence of the FITC-labeled pump; we confirmed that K(+) substantially decreased the fluorescence. Interestingly, Ba(2+) did shift the K(+) dose-response curve. Ethane diamine inhibited Mn(2+) stimulation of pNPPase (as well as K(+) and Mg(2+) stimulation) but did not shift the 50% inhibitory concentration (IC(50)) for the Mn(2+)-induced fluorescence change of FITC, though it did shift the IC(50) for the K(+)-induced change. These results suggest that the Mn(2+)-induced fluorescence change is not due to Mn(2+) binding at the transport site. The drawbacks of models in which Mn(2+) stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn(2+) stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.


Assuntos
4-Nitrofenilfosfatase/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , 4-Nitrofenilfosfatase/antagonistas & inibidores , Animais , Bário/metabolismo , Sítios de Ligação , Cães , Etilenodiaminas/farmacologia , Fluoresceína-5-Isotiocianato , Rim/enzimologia , Magnésio/metabolismo , Manganês/metabolismo , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Estrôncio/farmacologia
20.
Biochim Biophys Acta ; 1768(7): 1777-83, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17540337

RESUMO

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).


Assuntos
4-Nitrofenilfosfatase/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/fisiologia , Membrana Celular/enzimologia , Lipídeos/farmacologia , Magnésio/metabolismo , 4-Nitrofenilfosfatase/antagonistas & inibidores , 4-Nitrofenilfosfatase/sangue , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Química Encefálica , ATPases Transportadoras de Cálcio/sangue , Bovinos , Ativação Enzimática , Fosfatos/farmacologia , Fosfatidilcolinas/farmacologia , Potássio/farmacologia , Suínos , Vanadatos/farmacologia
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