title: Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies.
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.
authors.affiliation: Departamento de Química, Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil.
authors.forename: Marcela Cristina
authors.initials: MC
authors.lastname: de Moraes
authors.forename: Carmen L
authors.initials: CL
authors.lastname: Cardoso
authors.forename: Quezia B
authors.initials: QB
authors.lastname: Cass
mesh_terms: D000327:Adsorption; D000818:Animals; D001681:Biological Assay / Q000295:instrumentation*; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation*; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000032:analysis / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D011683:Purine-Nucleoside Phosphorylase / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D015203:Reproducibility of Results; D012550:Schistosoma mansoni / Q000201:enzymology*; D012556:Schistosomicides / Q000008:administration & dosage / Q000032:analysis / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation*
chemical_list: D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D012556:Schistosomicides; D011683:Purine-Nucleoside Phosphorylase