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Resistance of Nucleoside Analogues to HPRT-catalyzed pyrophosphorolysis
Because of its phosphorolysis activity, HGPRT can represent a threat for therapeutic efficacy of Nucleoside Analogues. NOVOCIB proposes HGPRT Assay Services which consist in treating a monophosphorylated nucleoside analogue by HGPRT with PPi. Pyrophosphorolysis is checked by HPLC analysis. Positive control is provided by IMP pyrophosphorolysis.
This pyrophosphorolysis assay service consists in treating a monophosphorylated Nucleotide Analogue* by HGPRT enzyme and evaluating their resistance to pyrophosphorolysis by human recombinant HGPRT.
* Monophosphorylated analogue can be of obtained from the phosphorylation of the corresponding nucleoside by Nucleoside Kinase (click here for details).
Because of its phosphorolysis activity, HGPRT can represent a threat for therapeutic efficacy of Nucleoside Analogues. NOVOCIB proposes HGPRT Assay Services which consist in treating a monophosphorylated nucleoside analogue by HGPRT with PPi. Pyrophosphorolysis is checked by HPLC analysis. Positive control is provided by IMP pyrophosphorolysis.
This pyrophosphorolysis assay service consists in treating a monophosphorylated Nucleotide Analogue* by HGPRT enzyme and evaluating their resistance to pyrophosphorolysis by human recombinant HGPRT.
* Monophosphorylated analogue can be of obtained from the phosphorylation of the corresponding nucleoside by Nucleoside Kinase (click here for details).
Hypoxanthine-guanine Phosphoribosyltransferase Cleaving Activity (in vitro)
Aim: Screening of compounds for their resitance to pyrophosphorolysis catalyzed by HGPRT
HGPRT enzyme: The enzyme used in this assay is a human recombinant HGPRT, cloned by NOVOCIB from human cells, expressed in E. coli, and produced and purified by NOVOCIB (click here for details). HGPRT purity and activity are controlled before running every assay.
HPLC analysis: The products derived from the pyrophosphorolysis of a nucleoside analogue by Human HGPRT are identified and quantified by HPLC, in comparison with positive control, using inosine monophosphate (IMP).
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| The enzymatic activity (reverse reaction) of human recombinant HGPRT was confirmed by ion-pair HPLC analysis (Agilent 1100 series, Zorbax C18plus) as shown by time-dependent decrease in IMP (blue: 0 min; red: 30 min; green: 3 h; pink: 24h; incubation at 37°C). To avoid forward reaction, hypoxanthine formed due to IMP pyrophosphorolysis was converted to uric acid due to added XDH. |
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