Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies.
Anal Bioanal Chem
; 405(14): 4871-8, 2013 May.
Article
em En
| MEDLINE
| ID: mdl-23535739
ABSTRACT
The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 µmol L (-1) and 164 ± 13.4 µmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Schistosoma mansoni
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Esquistossomicidas
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Espectrofotometria Ultravioleta
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Bioensaio
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Purina-Núcleosídeo Fosforilase
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Avaliação Pré-Clínica de Medicamentos
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Anal Bioanal Chem
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Brasil