Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Más filtros











Base de datos
Tipo de estudio
Intervalo de año de publicación
1.
Artículo en Chino | MEDLINE | ID: mdl-21970105

RESUMEN

OBJECTIVE: To clone and express adenine phosphoribosyltransferase gene of Schistosoma japonicum, and analyze its stage-specific transcription and expression at different developmental stages of S. japonicum. METHODS: Specific primers were designed according to the reported EST sequence of SjAPRT1 gene (GenBank Accession No. AAW24796). RT-PCR was used to investigate the differential transcription of SjAPRT1 gene during the developmental stages. The gene was cloned into pET28a(+) plasmid. The recombinant plasmid rSjAPRT1/pET28a(+) was transformed into E. coli BL21(DE3) and induced with IPTG. The recombinant protein was purified with Ni-NTA resin and analyzed by SDS-PAGE. The purified protein was used to immune New Zealand white rabbits to obtain the antiserum. Western blotting was used to investigate the immunogenicity and the differential expression of rSjAPRT1 at different developmental stages. RESULTS: RT-PCR result showed that the specific bands were detected in eggs, cercariae, schistosomula, and adult worms (561 bp). Western blotting analysis showed that the recombinant protein (rSjAPRT1, about Mr 25 000) existed in eggs, schistosomula and adult worms. The recombinant protein was recognized by pooled sera of infected rabbits. CONCLUSION: The recombinant protein (rSjAPRT1) shows specific immunoreactivity, and is detected in the stage of eggs, schistosomula, and adult worms.


Asunto(s)
Adenina Fosforribosiltransferasa/genética , Proteínas del Helminto/genética , Schistosoma japonicum/genética , Adenina Fosforribosiltransferasa/inmunología , Adenina Fosforribosiltransferasa/metabolismo , Animales , Clonación Molecular , Expresión Génica , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Conejos , Schistosoma japonicum/enzimología , Schistosoma japonicum/inmunología
2.
Mol Biochem Parasitol ; 134(1): 43-51, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-14747142

RESUMEN

The subcellular location of a protein is a critical factor in its physiological function and an important consideration in therapeutic paradigms that target the protein. Because Leishmania donovani cannot synthesize purine nucleotides de novo, they rely predominantly upon therapeutically germane phosphoribosyltransferase (PRT) enzymes, hypoxanthine-guanine PRT (HGPRT), adenine PRT (APRT), and xanthine PRT (XPRT), for purine acquisition from the host. Previous studies have shown that the L. donovani HGPRT is localized to the glycosome, a fuel-metabolizing microbody that is unique to kinetoplastid parasites [J. Biol. Chem. 273 (1998) 1534]. The sequences of the other two PRTs indicate that XPRT, but not APRT, possesses a COOH-terminal tripeptide that mediates protein targeting to the glycosome. To determine definitively the intracellular milieu of APRT and XPRT, polyclonal antibodies were raised to each recombinant protein. APRT and XPRT were then shown by immunofluorescence to be localized to the cytosol and glycosome, respectively. The glycosomal milieu for XPRT was also verified by immunoelectron microscopy. Amputation of the glycosomal targeting signal from XPRT resulted in protein mislocalization to the cytosol, but the cytosolic xprt was still functional with respect to purine salvage. These studies establish that APRT is cytosolic and XPRT, like the homologous HGPRT, is glycosomal and demonstrate that a mutant xprt protein that mislocalizes to the cytosol is still functional and supports parasite viability.


Asunto(s)
Adenina Fosforribosiltransferasa/metabolismo , Leishmania donovani/enzimología , Pentosiltransferasa/metabolismo , Adenina Fosforribosiltransferasa/inmunología , Animales , Citosol/enzimología , Genes Protozoarios , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/ultraestructura , Microcuerpos/enzimología , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Mutación , Pentosiltransferasa/genética , Pentosiltransferasa/inmunología , Señales de Clasificación de Proteína/genética , Transporte de Proteínas/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Xantina/metabolismo
3.
Enzyme ; 37(4): 182-8, 1987.
Artículo en Inglés | MEDLINE | ID: mdl-2440671

RESUMEN

We have studied adenine phosphoribosyltransferase (APRT) in the hemolysates from the families of 2,8-dihydroxyadenine urolithiasis associated with partial deficiency of APRT (the Japanese type) and complete deficiency of APRT (the null type). The APRT in the control subjects was found to be heat-stable at the physiological concentration of phosphoribosylpyrophosphate (PRPP), which was close to the value of its Km for PRPP. The APRT in the Japanese type showed 10 times higher Km values for PRPP and needed a comparably increased level of PRPP for stability in vitro. No change in red cell PRPP was found in the Japanese type of APRT deficiency. The content of APRT enzyme protein was decreased in the hemolysates of the Japanese type, probably due to its lability at the level of PRPP present in the cells. The heterozygote of the null type also had labile enzyme molecules at the physiological PRPP concentration.


Asunto(s)
Adenina Fosforribosiltransferasa/deficiencia , Adenina/análogos & derivados , Pentosiltransferasa/deficiencia , Cálculos Urinarios/genética , Adenina/análisis , Adenina Fosforribosiltransferasa/sangre , Adenina Fosforribosiltransferasa/inmunología , Preescolar , Eritrocitos/enzimología , Femenino , Heterocigoto , Calor , Humanos , Cinética , Persona de Mediana Edad , Linaje , Fosforribosil Pirofosfato/sangre , Cálculos Urinarios/enzimología
4.
Mutat Res ; 160(1): 61-9, 1986 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-3951457

RESUMEN

2,6-Diaminopurine(DAP)-resistant mutants have been isolated from mouse lymphoma 5178Y TK+/TK- heterozygotes. In the presence of 50 microM DAP, two colony types were isolated. Small colonies contained 50% wild-type adenine phosphoribosyl transferase (APRT) activity (partial mutants), whereas large colonies have undetectable levels of APRT (aprt- mutants). aprt- mutants could be isolated following mutagenesis with ICR-191 or EMS from the partial mutants. Southern blot analysis of EcoRI digested wild-type DNA using a 3.1 kb mouse aprt genomic probe indicated sequence polymorphism at one or both EcoRI sites flanking the allele. Southern blot analysis of one of the partial mutants and one ICR-induced aprt- mutant (single step) indicated that both strains were hemizygous at the APRT locus. Such stable hemizygous strains would be useful in short-term mutagen tests.


Asunto(s)
Adenina Fosforribosiltransferasa/genética , Leucemia L5178/genética , Leucemia Experimental/genética , Pentosiltransferasa/genética , 2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacología , Adenina Fosforribosiltransferasa/deficiencia , Adenina Fosforribosiltransferasa/inmunología , Animales , Línea Celular , Resistencia a Medicamentos , Genes , Heterocigoto , Inmunoensayo , Leucemia L5178/enzimología , Ratones , Mutágenos , Mutación , Hibridación de Ácido Nucleico , Timidina Quinasa/genética
5.
Mol Cell Biol ; 5(10): 2662-8, 1985 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-3837181

RESUMEN

Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) pseudorevertant cell lines were isolated under selective conditions requiring adenine salvage for survival; yet they were found to be deficient in measurable APRT activity and resistant to the purine analog 2'6'-diaminopurine (DAP) (M.S. Turker, J. A. Tischfield, P. Rabinovitch, P.J. Stambrook, J.J. Trill, A.C. Smith, C.E. Ogburn, and G.M. Martin, manuscript in preparation). Adenine salvage was examined in two APRT pseudorevertant cell lines, their two APRT homozygous deficient parental cell lines, and a genotypic APRT revertant cell line (i.e., one with measurable APRT activity and DAP sensitivity). Adenine accumulation was observed in both revertant phenotypes and was demonstrated by high-performance liquid chromatography to be linked with adenine metabolism. The ability to salvage adenine declined substantially in the pseudorevertant cell lines when they were removed from selective media containing inhibitors of de novo 5'-AMP synthesis (alanosine and azaserine); for one pseudorevertant cell line this decline was accelerated by the addition of DAP to the medium. The readdition of alanosine or azaserine to the growth medium of the pseudorevertant lines induced adenine salvage to its previous levels. An APRT-like cross-reacting material was found in the pseudorevertant cell lines, although its relationship to adenine salvage is unknown. A low level of constitutive adenine salvage was found in the parental APRT-deficient lines, and it was also possible to induce adenine salvage in these cell lines. These findings suggest a novel regulatory mechanism for adenine salvage.


Asunto(s)
Adenina Fosforribosiltransferasa/deficiencia , Adenina/metabolismo , Pentosiltransferasa/deficiencia , 2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacología , Adenina Fosforribosiltransferasa/inmunología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Azaserina/farmacología , Línea Celular , Reacciones Cruzadas , Resistencia a Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Cariotipificación , Ratones , Mycoplasma/análisis
6.
Mol Cell Biol ; 2(3): 250-7, 1982 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-7110133

RESUMEN

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.


Asunto(s)
Adenina Fosforribosiltransferasa/genética , Pentosiltransferasa/genética , Adenina Fosforribosiltransferasa/inmunología , Adenina Fosforribosiltransferasa/metabolismo , Animales , Células Cultivadas , Células Clonales/enzimología , Cricetinae , Cricetulus , Reacciones Cruzadas , Femenino , Regulación de la Expresión Génica , Genes , Humanos , Células Híbridas/enzimología , Células L/enzimología , Ratones , Mutación , Ovario , Transformación Genética
7.
Hum Genet ; 57(4): 404-10, 1981.
Artículo en Inglés | MEDLINE | ID: mdl-7286981

RESUMEN

In order to study the biochemical basis of a complete deficiency of adenine phosphoribosyl transferase (APRT) the enzyme was purified to homogeneity, its properties were characterized, and antibodies raised. The enzyme is indirectly involved in adenine uptake. Apparently, by forming AMP the internal concentration of adenine is kept low allowing it diffusion. The same APRT is present in various tissues as was revealed by antibody inactivations employing anti-erythrocyte APRT as well as by direct enzyme assays in cells from the APRT deficient patient. In vitro cultured fibroblasts derived from this patient had less than 0.02% enzyme activity. No cross-reacting material was found in erythrocytes obtained from an APRT deficient child.


Asunto(s)
Adenina Fosforribosiltransferasa/deficiencia , Adenina Fosforribosiltransferasa/metabolismo , Pentosiltransferasa/deficiencia , Pentosiltransferasa/metabolismo , Adenina , Adenina Fosforribosiltransferasa/inmunología , Reacciones Antígeno-Anticuerpo , Reacciones Cruzadas , Eritrocitos/inmunología , Femenino , Humanos , Masculino , Linaje , Distribución Tisular
8.
Humangenetik ; 30(1): 23-34, 1975 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-1184004

RESUMEN

Male New Zealand White rabbits were immunized with human adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which were purified about 2000-fold and 800-fold, respectively, from erythrocytes by DEAE-cellulose chromatography, ammonium sulfate precipitation and preparative polyacrylamide gel electrophoresis. Specific immunoprecipitations of APRT and HGPRT were achieved with the antisera that were obtained and by using polyethylene glycol as a substitute for goat anti-(rabbit) gamma globulin. The activities of the human forms of these enzymes, whether from red blood cells or from cultured cells, were almost completely eliminated under the conditions of immunoprecipitation used. Little or no reduction of APRT and HGPRT activities from mouse and Chinese hamster cells was observed. This discriminatory capacity of the antisera was successfully used for the identification of human APRT and HGPRT in human-mouse and human-hamster cell hybrids using the immunoprecipitation reaction.


Asunto(s)
Adenina Fosforribosiltransferasa/análisis , Células Híbridas/enzimología , Hipoxantina Fosforribosiltransferasa/análisis , Pentosiltransferasa/análisis , Adenina Fosforribosiltransferasa/inmunología , Animales , Cricetinae , Eritrocitos/enzimología , Femenino , Fibroblastos/enzimología , Humanos , Hipoxantina Fosforribosiltransferasa/inmunología , Sueros Inmunes , Masculino , Ratones , Pruebas de Precipitina , Conejos/inmunología , Especificidad de la Especie
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA