RESUMEN

Aqueous two-phase micellar system (ATPMS), as an alternative liquid-liquid extraction technique, has been extensively exploited for the precise separation or large-scale concentration of biomolecules. In this article, a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a Copper-chelated Triton X-114 (TX-Cu(II)) into an aqueous solution of hydrophobically modified ethylene oxide polymer (HM-EO). Phase diagram of the HM-EO/TX-Cu(II) system was measured, and the partitioning behavior of model proteins (YND, BSA, lysozyme) were studied by using this new system. The addition of HM-EO can result in formation of the micellar network in the micelle-rich phase, making the phase separation easier and stabler. In addition, the extractive performance of ATPMS was enhanced due to the existence of the mixed micelles composed by HM-EO and Cu(II)-chelated TX. It was found in the partitioning experiments that the hexahistidine-tagged Yeast 3',5'-bisphosphate nucleotidase (YND) was selectively extracted into the micelle-rich phase, while the histidine-poor proteins (BSA and lysozyme) remained in the micelle-poor phase. Finally, HM-EO/TX-Cu(II) was used directly to process the fermentation broth. The target protein, YND could be recovered from the cell lysate with a recovery yield of 49.23% and purification factor of 2.63. The results indicated that the new affinity-based HM-EO/TX-Cu(II) system had high partitioning performance which is promising for effectively separation of the histidine-tagged proteins.

Asunto(s)

Proteínas Fúngicas/aislamiento & purificación , Micelas , Nucleotidasas/aislamiento & purificación , Levaduras/enzimología , Cromatografía Líquida de Alta Presión , Óxido de Etileno , Proteínas Fúngicas/análisis , Extracción Líquido-Líquido , Espectrometría de Masas , Nucleotidasas/análisis

RESUMEN

Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can be also generated by TET enzymes. All of the aforementioned modifications seem to play some regulatory roles. We applied isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of the 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. Analyses of DNA extracted from matched human samples showed that the 5-(hydroxymethyl)-2'-deoxycytidine level was 5-fold lower in colorectal carcinoma tumor in comparison with the normal one from the tumor's margin; also 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine were lower in colorectal carcinoma tissue (ca. 2.5- and 3.5-fold, respectively). No such differences was found for 2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxyuridine. The presented methodology is suitable for fast, accurate, and complex evaluation of an array of endogenously generated DNA deoxynucleosides modifications. This novel technique could be used for monitoring of cancer and other diseases related to oxidative stress, aberrant metabolism, and environmental exposure. Furthermore, the fully automated two-dimensional separation is extremely useful for analysis of material containing a considerable amount of coeluting interferents with mass-spectrometry-based methods.

Asunto(s)

Biomarcadores/análisis , Cromatografía Líquida de Alta Presión/métodos , Nucleotidasas/análisis , Espectrometría de Masas en Tándem/métodos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/análisis , 5-Metilcitosina/metabolismo , Animales , Encéfalo/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , ADN/aislamiento & purificación , ADN/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/análisis , Desoxicitidina/metabolismo , Humanos , Marcaje Isotópico , Oxigenasas de Función Mixta/metabolismo , Nucleotidasas/aislamiento & purificación , Reproducibilidad de los Resultados , Porcinos , Timo/metabolismo

RESUMEN

Affinity chromatography is one of the most popular methods for protein purification. Each tag method has its advantages and disadvantages, and combination of different tags and developing of new tags had been proposed and performed. Yeast 3',5'-bisphosphate nucleotidase, also known as HAL2, hydrolyzes 3'-phosphoadenosine 5'-phosphate (PAP) with submicromolar Km, which indicated the tight interactions between HAL2 and PAP. In order to explore the feasibility of HAL2 as a protein purification affinity tag, HAL2 was further characterized with PAP as substrate. Results demonstrated that KmPAP and kcatPAP were ∼0.3µM and ∼11s(-)(1), respectively. Kd for PAP was 0.008µM in the presence of Ca(2+). pH was also found to affect interactions between HAL2 and PAP, with tightest binding (Kd∼8nM) at pH 7.5 and 8. The purification protocol was rationally designed based on nanomolar affinity to PAP agarose in the presence of Ca(2+), which could satisfy the metal requirement for PAP binding, prevent hydrolysis of immobilized PAP and could be chelated by ethylene glycol tetraacetic acid (EGTA) for elution. A series of expression vectors were further constructed and Escherichia coli adenosine 5'-phosphosulfate kinase (APSK) was prokaryotically expressed, purified and characterized. Ready to use expression vector with eight commonly used restriction enzyme recognition sites in multiple cloning site was subsequently constructed. By comparing with current popular tags, HAL2 was found to be an efficient and economical tag for prokaryotic protein expression and purification.

Asunto(s)

Vectores Genéticos/genética , Nucleotidasas/genética , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/enzimología , Adenosina Difosfato/metabolismo , Clonación Molecular/métodos , Escherichia coli/enzimología , Escherichia coli/genética , Hidrólisis , Nucleotidasas/aislamiento & purificación , Nucleotidasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética

RESUMEN

The venom of the snake Bothrops asper causes muscle necrosis, pain and inflammation. This venom contains myotoxins which cause an increase in intracellular Ca(2+) concentration and release of K(+) and ATP from myotubes. ATP is a key danger molecule that triggers a variety of reactions, including activation of the innate immune response. Here, using ATP-luciferase bioluminescence imaging technique, we show for the first time in vivo, that the purified myotoxins induce rapid release of ATP, whilst the complete venom of B. asper does at a very small extent. This apparent contradiction is explained by the finding that the venom contains powerful nucleotidases that in vivo convert ATP into ADP, AMP and Adenosine. These findings indicate that high concentrations of adenosine are generated by the double action of the venom and provide the experimental basis to the suggestion that in situ generated adenosine plays an important role in envenomation via its hypotensive, paralyzing and anti-coagulant activities.

Asunto(s)

Adenosina Trifosfato/metabolismo , Venenos de Crotálidos/enzimología , Fosfolipasas A2 Grupo II/farmacología , Nucleotidasas/farmacología , Proteínas de Reptiles/farmacología , Adenosina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Fosfolipasas A2 Grupo II/química , Fosfolipasas A2 Grupo II/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL , Nucleotidasas/química , Nucleotidasas/aislamiento & purificación , Proteínas de Reptiles/química , Proteínas de Reptiles/aislamiento & purificación

RESUMEN

The final step in RNA degradation is the hydrolysis of RNA fragments five nucleotides or less in length (nanoRNA) to mononucleotides. In Escherichia coli this step is carried out by oligoribonuclease (Orn), a DEDD-family exoribonuclease that is conserved throughout eukaryotes. However, many bacteria lack Orn homologs, and an unrelated DHH-family phosphoesterase, NrnA, has recently been identified as one of the enzymes responsible for nanoRNA degradation in Bacillus subtilis. To understand its mechanism of action, B. subtilis NrnA was purified and crystallized at room temperature using the hanging-drop vapor-diffusion method with PEG 4000, PEG 3350 or PEG MME 2000 as precipitant. The crystals belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 50.62, b = 121.3, c = 123.4 Å, α = 90, ß = 91.31, γ = 90°.

Asunto(s)

Bacillus subtilis/enzimología , Nucleotidasas/química , Cristalización , Cristalografía por Rayos X , Nucleotidasas/aislamiento & purificación

RESUMEN

In this study, we report isolation of a phosphatase gene designated GhHL1 from cotton and its functional characterization. GhHL1 transcripts were detected in all cotton tissues examined. Southern blotting analysis indicated that it exists in multiple-copies. Biochemical analysis showed that GhHL1 was Mg2+-dependent and cation-sensitive. Purified recombinant GhHL1 protein dephosphorylated both 3',5'-bisphosphate nucleotide and inositol 1,4-bisphosphate, demonstrating dual 3',5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. Overexpression of GhHL1 complemented yeast hal2 mutant and enhanced yeast growth under elevated NaCl or LiCl, showing a role in salt tolerance associated with ionic stress response. Taken together, these results show that GhHL1 is a functional and good candidate gene, which might be used to improve salt tolerance in plants.

Asunto(s)

Gossypium/enzimología , Nucleotidasas/química , Monoéster Fosfórico Hidrolasas/química , Secuencia de Bases , Clonación Molecular , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Nucleotidasas/genética , Nucleotidasas/aislamiento & purificación , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Cloruro de Sodio , Temperatura

RESUMEN

Nucleoside 5'-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25 degrees C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5'-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA(Yi)) of the Y. intermedia ushA gene (ushA(Yi)) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshA(Ec)). Therefore, UshA(Ec) was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA(Ec) and UshA(Yi) behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with k(cat)/K(m) ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA(Ec) conformers and the respective homology models constructed for UshA(Yi), and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.

Asunto(s)

Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Nucleotidasas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Azúcares de Uridina Difosfato/metabolismo , Yersinia/enzimología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Escherichia coli/química , Escherichia coli/genética , Expresión Génica , Cinética , Datos de Secuencia Molecular , Nucleotidasas/química , Nucleotidasas/genética , Nucleotidasas/aislamiento & purificación , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Ratas , Especificidad por Sustrato , Azúcares de Uridina Difosfato/química , Yersinia/química , Yersinia/genética

RESUMEN

NADP phosphatase (NADPase) is an enzyme that converts NADP+ into NAD+ through dephosphorylation of NADP+, and is considered to be one of the possible candidates for regulation of the NAD+/NADP+ balance in vivo. In order to obtain an intrinsic NADPase, the NADP+-degrading activity in a membrane-free cell extract of a Gram-positive bacterium, Arthrobacter sp. strain KM, was first assessed and demonstrated to be mainly achieved through the NADPase reaction, indicating NADPase is essential for degradation of NADP+ and therefore for regulation of the NAD+/NADP+ balance in cytosol. Then, the isolation of cytosolic NADPase was attempted using NADP+ as a substrate. Two NADPase isozymes, designated as NADPases I and II, were purified from the cell extract of the bacterium, and were indicated to be the sole cytosolic NADPases regulating the balance of NAD+/NADP+. NADPases I and II are homodimers of 32 and 30 kDa subunits, respectively, and most active at pH 7-8. The N-terminal amino acid sequences of the two enzymes are similar to each other. Among the biological substrates tested, both enzymes showed the highest activity toward NADP+ and NADPH. AMP, ADP, and pyridoxal 5'-phosphate were also dephosphorylated, but to lower extents. Comparison of the features of NADPases I and II with those of other acid phosphatases possessing NADPase activity suggested that NADPases I and II are novel enzymes participating in regulation of the NAD+/NADP+ balance in the cytosol.

Asunto(s)

Arthrobacter/enzimología , Citoplasma/enzimología , Isoenzimas/metabolismo , NADP/metabolismo , NAD/metabolismo , Nucleotidasas/metabolismo , Fosfatasa Ácida/fisiología , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Secuencia de Aminoácidos , Dimerización , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Nucleotidasas/química , Nucleotidasas/aislamiento & purificación , Subunidades de Proteína/química , Fosfato de Piridoxal/metabolismo , Especificidad por Sustrato

RESUMEN

YfkN isolated from the culture supernatant of Bacillus subtilis in the exponential phase of growth is a protein of 143.5 kDa that derives from a putative large precursor of 159.6 kDa processed at both the N- and C-terminal ends. Pulse-chase experiments indicated that the release occurs slowly with a half-time longer than 30 min, suggesting that the event is coupled with wall turnover. YfkN exhibits 2',3' cyclic nucleotide phosphodiesterase, 2' (or 3') nucleotidase and 5' nucleotidase activities. In vitro the protein is reduced by subtilisin digestion to a shorter polypeptide (68 kDa), displaying phosphodiesterase activity but devoid of any 5'nucleotidase activity. This proteolytic processing led us to localize the potential active sites of the various nucleotidase activities. When bacteria were grown in low phosphate medium, the exocellular production of the enzyme was enhanced, suggesting that it plays a role in phosphate metabolism. Comparison with nucleotidase databases suggests that yfkN resulted from gene fusion.

Asunto(s)

2',3'-Nucleótido Cíclico Fosfodiesterasas/aislamiento & purificación , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Bacillus subtilis/enzimología , Nucleotidasas/aislamiento & purificación , Nucleotidasas/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Cinética , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Subtilisina/metabolismo

RESUMEN

The fruit body of shiitake (Lentinus edodes) produces two acid nucleases, nuclease Le1 and nuclease Le3, both of which are thought to be candidates for the enzyme that produces a flavorful substance, 5'-GMP, and the primary structure of one of the nucleases, nuclease Le1, has been analyzed by both protein chemistry and gene cloning [Biosci. Biotechnol. Biochem. 64, 948-957 (2000)]. In this study the amino acid sequence of nuclease Le3 was analyzed by protein chemistry and gene cloning. Nuclease Le3 is a glycoprotein that contains 280 amino acid residues, and the molecular mass of the protein moiety of nuclease Le3 is 31,045. The nucleotide sequence of the cDNA and genomic DNA encoding nuclease Le3 revealed the presence of an 18-residue putative signal peptide. Nuclease Le3 contains 170, 108, and 98 amino acid residues that are identical to residues of nuclease Le1, nuclease P1, and nuclease S, respectively. The amino acid residues involved in coordination with Zn2+ atoms in nuclease P1 are all conserved in nuclease Le3. Nuclease Le3 contains 9 half-cystine residues, and 7 of them are located in the same positions as in nuclease Le1.

Asunto(s)

Nucleotidasas/aislamiento & purificación , Nucleotidasas/metabolismo , Hongos Shiitake/enzimología , Hongos Shiitake/genética , Secuencia de Aminoácidos , Secuencia de Bases , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Nucleotidasas/química , Nucleotidasas/genética , Filogenia , ARN/genética , ARN/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

Cells that depend on oxygen for survival constantly produce reactive oxygen species that attack DNA to produce a variety of lesions, including single-strand breaks with 3'-blocking groups such as 3'-phosphate and 3'-phosphoglycolate. These 3'-blocking ends prevent the activity of DNA polymerase and are generally removed by DNA repair proteins with 3'-diesterase activity. We report here the purification and partial characterization of a 45 kDa protein from Schizosaccharomyces pombe total extract based on the ability of this protein to process bleomycin- or H(2)O(2)-damaged DNA in vitro to allow DNA repair synthesis by DNA polymerase I. Further analysis revealed that the 45 kDa protein removes 3'-phosphate ends created by the Escherichia coli fpg AP lyase following the incision of AP site but is unable to process the 3'-alpha,beta unsaturated aldehyde generated by E. coli endonuclease III. The protein cannot cleave DNA bearing AP sites, suggesting that it is not an AP endonuclease or AP lyase. We conclude that the 45 kDa protein purified from S. pombe is a DNA 3'-phosphatase.

Asunto(s)

Nucleotidasas/química , Nucleotidasas/aislamiento & purificación , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/aislamiento & purificación , Cationes Bivalentes/farmacología , Reacciones Cruzadas , Daño del ADN , Reparación del ADN , Humanos , Peróxido de Hidrógeno/toxicidad , Sueros Inmunes/metabolismo , Magnesio/farmacología , Nucleotidasas/metabolismo , Fosfatos/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/inmunología , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo

RESUMEN

Previously, we isolated and characterized the gene encoding the 3'-Nucleotidase/Nuclease (Ld3'NT/NU) from the human pathogen, Leishmania donovani. This unique cell surface enzyme has been shown to be involved in the salvage of host-derived purines, which are essential for the survival of this important protozoan parasite. In this report, we assessed whether the 3'-Nucleotidase/Nuclease was conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that a Ld3'NT/NU gene homolog was present in each of the visceral and cutaneous Leishmania species tested (i.e. isolates of L. donovani, L. infantum, L. tropica, L. major and L. mexicana, respectively). Further, results of colorimetric assays using 3'-adenosine monophosphate as substrate demonstrated that each of these organisms also expressed significant levels of 3'-nucleotidase enzyme activity. In addition, we showed that a Ld3'NT/NU gene homolog was expressed in each of these Leishmania species as a > 40 kDa 3'-nucleotidase enzyme activity. A Ld3'NT/NU gene homolog was also identified in two Crithidia species (C. fasciculata and C. luciliae) and Leptomonas seymouri but was only marginally detectable in Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens. Cumulatively, results of this study showed that an Ld3'NT/NU homolog was conserved amongst pathogenic Leishmania sp. which suggests that this enzyme must play an critical role in purine salvage for all members of this group of human pathogens.

Asunto(s)

Leishmania donovani/química , Trypanosoma/química , Animales , Southern Blotting , Western Blotting , ADN Protozoario/análisis , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Epítopos , Humanos , Nucleotidasas/química , Nucleotidasas/aislamiento & purificación , Oligonucleótidos/metabolismo , Unión Proteica , Purinas/química , ARN Protozoario/análisis

RESUMEN

Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP >> UTP. These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Portadoras/metabolismo , Nucleotidasas/metabolismo , Nucleótidos de Purina/metabolismo , Retina/química , Retina/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Fraccionamiento Celular , Cromatografía en Agarosa , Clonación Molecular , Escherichia coli/enzimología , Escherichia coli/genética , GTP Fosfohidrolasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Hidrólisis , Péptidos y Proteínas de Señalización Intracelular , Cinética , Nucleotidasas/genética , Nucleotidasas/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificación , Plásmidos/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Ribonucleasas/aislamiento & purificación

RESUMEN

Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.

Asunto(s)

Arabidopsis/genética , Nucleotidasas/genética , Hojas de la Planta/genética , Proteínas de Plantas/genética , Tallos de la Planta/genética , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis , Asteraceae/genética , Asteraceae/metabolismo , Secuencia de Bases , Northern Blotting , Desoxirribonucleasas/metabolismo , Datos de Secuencia Molecular , Nucleotidasas/aislamiento & purificación , Nucleotidasas/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Tallos de la Planta/metabolismo , Ribonucleasas/metabolismo , Alineación de Secuencia

RESUMEN

A lectin isolated from the roots of the legume, Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date. This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands. This lectin-nucleotide phosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes. LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia. These properties suggest that this protein may play a role in the rhizobium-legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant.

Asunto(s)

Apirasa/metabolismo , Fabaceae/enzimología , Lectinas/metabolismo , Nucleotidasas/metabolismo , Proteínas de Plantas , Plantas Medicinales , Secuencia de Aminoácidos , Apirasa/aislamiento & purificación , Quitina/metabolismo , Clonación Molecular , Fabaceae/microbiología , Técnica del Anticuerpo Fluorescente , Sueros Inmunes/inmunología , Sueros Inmunes/farmacología , Lectinas/aislamiento & purificación , Datos de Secuencia Molecular , Nucleotidasas/química , Nucleotidasas/aislamiento & purificación , Monoéster Fosfórico Hidrolasas/metabolismo , Lectinas de Plantas , Raíces de Plantas/enzimología , Polisacáridos/metabolismo , Unión Proteica , Rhizobium/metabolismo , Especificidad por Sustrato

RESUMEN

Soluble broad spectrum 5'-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH4)2SO4 precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min-1 mg-1. SDS-PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg2+ and Mn2+ were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, Pi was a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[alpha, beta-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms.

Asunto(s)

Nucleotidasas/química , Nucleotidasas/aislamiento & purificación , Semen/enzimología , Adenosina/biosíntesis , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Estabilidad de Enzimas , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/aislamiento & purificación , Masculino , Metales/farmacología , Peso Molecular , Nucleotidasas/efectos de los fármacos , Fosfatos/metabolismo , Fosfatos/farmacología , Semen/química , Solubilidad , Especificidad por Sustrato , Temperatura

RESUMEN

Leishmania donovani and related trypanosomatid protozoa possess an externally oriented surface membrane enzyme capable of hydrolyzing both 3'-nucleotides and nucleic acids. By virtue of these activities, this 3'-nucleotidase/nuclease (3'-NT/Nu), previously shown to be analogous to fungal and plant class-I single-strand-specific nucleases, is thought to play a critical role in the salvage of purines, essential for the survival of these organisms. The 43-kDa 3'-NT/Nu was purified from L. donovani promastigotes and trypsin treated. Four of the released tryptic peptide fragments yielded amino-acid sequence information (Pept-1 to Pept-4) which provided the basis for the preparation of oligonucleotide primers used for PCR amplification of an approx. 300-bp DNA fragment. This fragment was cloned, sequenced and used to probe a genomic L. donovani cosmid library. Nucleotide sequence analysis of a 4.5-kb SmaI fragment, isolated from a cosmid clone, revealed an open reading frame (ORF) of 1434 nt encoding a 477-amino-acid protein. Pept-1 to Pept-4 were mapped onto the ORF-deduced protein sequence. Peptides corresponding to Pept-1 to Pept-4 were synthesized and used to immunize rabbits. The resulting anti-peptide antibodies recognized the 43-kDa protein on Western blots and immunoprecipitated the native 3'-nucleotidase activity from L. donovani membrane extracts. Further, the ORF-deduced protein shared significant sequence identity with the S1 and P1 fungal nucleases of Aspergillus oryzae and Penicillium citrinum, respectively. Cumulatively, these results demonstrated that the ORF corresponded to a gene for the L. donovani 3'-nucleotidase/nuclease. In Northern blots a nucleotide probe specific for the 3'-NT/Nu gene hybridized to a single 2.5-kb messenger RNA. Results of Southern blot analyses were consistent with the 3'-NT/Nu being encoded by a single copy gene. These data constitute the first report of the gene for this unique trypanosomatid surface membrane enzyme.

Asunto(s)

Membrana Celular/enzimología , Genes Protozoarios/genética , Leishmania donovani/genética , Nucleotidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Protozoario/análisis , Leishmania donovani/enzimología , Datos de Secuencia Molecular , Peso Molecular , Nucleotidasas/química , Nucleotidasas/aislamiento & purificación , Sistemas de Lectura Abierta/genética , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , ARN Protozoario/análisis , Alineación de Secuencia , Análisis de Secuencia

Asunto(s)

Hígado/enzimología , Nucleotidasas/metabolismo , Placenta/enzimología , Animales , Anticuerpos , Núcleo Celular/enzimología , Pollos/inmunología , Citosol/enzimología , Femenino , Humanos , Inmunodifusión , Lisosomas/enzimología , Mitocondrias Cardíacas/enzimología , Nucleotidasas/inmunología , Nucleotidasas/aislamiento & purificación , Embarazo

RESUMEN

A plasma membrane 3'-nucleotidase (3'-ribonucleotide phosphohydrolase, EC 3.1.3.6) having a apparent subunit molecular mass of 38 kDa but filtrable through a Centriprep-10 microconcentrator (Amicon) membrane was purified from Leishmania donovani promastigotes. The enzyme activity has an optimum at pH around 7.5. EDTA strongly inhibited the enzyme activity which was fully restored only by Co2+, from various metal ions added. Citrate ions, Zn2+ or dithiothreitol were also strongly inhibitory. The enzyme is apparently located on the inner side of the parasite plasma membrane. The substrate specificity and kinetics of the filtrable enzyme are similar to those of the non-filtrable outer-surface-membrane-bound 3'-nucleotidase reported by Gbenle and Dwyer (Gbenle, G.O. and Dwyer, D.M. (1992) Biochem. J. 285, 41-46). Therefore, it is suggested that both enzymes are implicated in the supply of nucleosides to the parasite.

Asunto(s)

Leishmania donovani/enzimología , Nucleotidasas/aislamiento & purificación , Animales , Membrana Celular/enzimología , Ditiotreitol/farmacología , Ácido Edético , Concentración de Iones de Hidrógeno , Cinética , Nucleotidasas/antagonistas & inhibidores , Nucleotidasas/química , Especificidad por Sustrato , Zinc/farmacología

RESUMEN

The parasitic protozoon Leishmania mexicana was examined for the presence of 3'-nucleotidase/nuclease using substrate SDS-PAGE. Two activities were detected: one with an apparent molecular mass of 40 kDa, and a doublet of 29/31 kDa. The two enzymes showed differences in their levels of expression in the two life-cycle stages of parasite examined: the 29/31 kDa doublet was detected at 60-fold higher levels in the pathogenic amastigote stage, but was also expressed by promastigotes, whereas the 40 kDa activity was only detected in promastigotes. Both were capable of hydrolysing a variety of substrates including 3'-AMP and poly(A). However, the 29/31 kDa form showed a broader, unique substrate specificity in that it was also capable of digesting double-stranded RNA and DNA.

Asunto(s)

Leishmania mexicana/enzimología , Nucleotidasas/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Animales , Femenino , Regulación Enzimológica de la Expresión Génica , Leishmania mexicana/crecimiento & desarrollo , Ratones , Ratones Endogámicos CBA , Peso Molecular , Nucleotidasas/metabolismo , Proteínas Protozoarias/metabolismo , Especificidad por Sustrato