Product Overview :
PNP Assay Kit provides an enzymatic tool allowing direct continuous spectrophotometric monitoring of PNP activity in a convenient 96-well plate format. In the assay, PNP activity is measured as a rate of production of hypoxanthine, which is directly oxidized by XDH enzyme with concomitant reduction of NAD+ to NADH measurable by absorbance at 340nm.
The assay is developed for measuring PNP activity in vitro or in cell lysates.
For maximal accuracy, the assays with cell lysates are run with and without inosine in parallel. The absorbance rate observed in the absence of inosine is used as blank and is subtracted from the absorbance rate measured in its presence.
Description :
Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is involved in salvage pathway of the purine metabolism. In the presence of inorganic phosphate PNP catalyzes the cleavage of the glycosidic bond of ribo- or deoxyribonucleosides (inosine, guanosine or their deoxyanalogues) to generate the purine base and ribose- or deoxyribose-1-phosphate. The reaction is reversible for natural substrates.
Purine nucleoside phosphorylase deficiency is a rare autosomal recessive metabolic disorder which results in accumulation of toxic metabolites in T-cell lymphocytes and severe immunodeficiency. In addition, purine nucleoside phosphorylase deficiency is associated with neurologic symptoms, including mental retardation and muscle spasticity, and increased risk of autoimmune disorders. Since most parasitic protozoan are obligate auxotrophs of purines and entirely depend therefore on their purine salvage pathways, PNP of apicomplexan parasites (P. falciparum and T. gondii) is currently explored as a potential target for drug development.
Storage :
Once dissolved, the reagents provided in the kit are not stable and should be stored on ice and used the day of preparation.
Kit Components :
The kit allows to perform 24 analysis in a time (8 samples in triplicate, 12 samples in duplicate).
A standard PNP Assay Kit contains:
one 2mL-tube "Cofactor 1" (DTT);
one 2mL-tube "Cofactor 2" (NAD);
one vial "XDH Enzyme";
one 15mL tube "Blank" (pre-filled with 10mL of "Reaction buffer");
one 15mL tube "Reaction mixture" (pre-filled with 50μL of 100mM inosine);
one tube of "Purified PNP" (human recombinant) to be used as a positive control;
one transparent 96-well plate.
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