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1.
Säo Paulo; s.n; 2001. 180 p. ilus, tab, graf.
Thèse Dans Portugais | LILACS | ID: lil-289807

Résumé

Este trabalho mostrou a purificação de uma apirase em extratos de tegumentos de vermes adultos de S. mansoni com razão de hidrólise de ADP/ATP aproximadamente 2. Análise da amostra por MALDI-TOF evidenciou a presença de helicase e carboxilesterase, sendo que a atividade de hidrólise de ADP ainda não foi descrita para estas proteínas. Através de ®Western blot¼ mostrou-se que a proteína purificada não tem epítopos reconhecíveis por anticorpo anti-apirase de batata, e que a imunoreatividade cruzada com apirase da família CD39 está presente na fração insolúvel que não foi utilizada no processo de purificação. Esta tese caracteriza também a utilização de ®primers¼ consenso degenerados na construção de bibliotecas de cDNA com PCR de baixa estringência...


Sujets)
Animaux , Souris , Antigènes/usage thérapeutique , Apyrase/isolement et purification , Clonage d'organisme , Banque de gènes , Schistosoma mansoni/immunologie , Schistosomiase/prévention et contrôle , Technique de Western , Électrophorèse sur gel de polyacrylamide , Réaction de polymérisation en chaîne
2.
Braz. j. med. biol. res ; 33(7): 725-9, July 2000. graf
Article Dans Anglais | LILACS | ID: lil-262672

Résumé

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.


Sujets)
ADP/métabolisme , Apyrase/métabolisme , Nucléotides/métabolisme , Solanum tuberosum/enzymologie , Apyrase/composition chimique , Apyrase/isolement et purification , Césium/composition chimique , Césium/métabolisme , Iode/composition chimique , Iode/métabolisme , Isoenzymes/composition chimique , Solanum tuberosum/composition chimique , Spectrométrie de fluorescence
3.
Ciênc. cult. (Säo Paulo) ; 47(3): 131-6, May-Jun. 1995. tab
Article Dans Anglais | LILACS | ID: lil-191370

Résumé

This review aimed at discussing the protocols used to characterize and to distinguish ATP diphosphohydrolases from other enzymes which can promote the degradation of ATP and ADP, since there is a confusion about the identily of this enzyme and ATPases.


Sujets)
Adénosine triphosphate , Apyrase , Adenosine triphosphatases/antagonistes et inhibiteurs , Adénosine triphosphate/isolement et purification , Apyrase/isolement et purification , Apyrase/métabolisme
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