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* Pricing updated September 23rd, 2011. Prices are subject to change without notice. Shipping charges are not included. ** One unit of hypoxanthine-guanine phosphoribosyl transferase converts 1 µmole of hypoxanthine to IMP per minute at pH 7.5 at 37°C, as measured by a coupled bacterial IMPDH (NovoCIB) enzyme system. Specific Activity: ≥ 0.8 U/mg protein. For other size, please contact us Click to order or to get further information |
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Human Recombinant HGPRT - Product description
Synonyms: Hypoxanthine phosphoribosyltransferase, HGPRTase, HPRT
NOVOCIB's human HGPRT is a recombinant protein of ca. 25kDa cloned by RT-PCR amplification of mRNA extracted from human hepatoma cells and expressed in E.coli. The sequence of the cloned HGPRT (accession number P00492) was confirmed by DNA sequencing (100% identity).
Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) is a purine salvage enzyme that catalyzes the reversible transfer of the 5-phosphoribosyl group between α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and a purine base (hypoxanthine or guanine) to form a purine nucleotide IMP or GMP. The reaction is reversible but forward reaction (nucleotide formation) is heavily favoured.
HGPRTase has been extensively investigated because defects within the human enzyme are associated with genetically inherited gouty arthritis and Lesch–Nyhan syndrome(1).
Since the hydrolysis of nucleotides analogues of inosine or guanosine by HPRT may limit their efficiency, NOVOCIB has developed an HGPRT enzymatic assay assay which consists in treating monophosphorylated Nucleoside Analogues by HGPRT and evaluating by HPLC their resistance to pyrophosphorolysis by human recombinant HGPRT.
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Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) is a purine salvage enzyme that catalyzes the reversible transfer of the 5-phosphoribosyl group between α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and a purine base (hypoxanthine or guanine) to form a purine nucleotide IMP or GMP. The reaction is reversible but forward reaction (nucleotide formation) is heavily favoured.
HGPRTase has been extensively investigated because defects within the human enzyme are associated with genetically inherited gouty arthritis and Lesch–Nyhan syndrome(1).
Since the hydrolysis of nucleotides analogues of inosine or guanosine by HPRT may limit their efficiency, NOVOCIB has developed an HGPRT enzymatic assay assay which consists in treating monophosphorylated Nucleoside Analogues by HGPRT and evaluating by HPLC their resistance to pyrophosphorolysis by human recombinant HGPRT.
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Storage: HGPRT enzyme is provided in lyophilized form and should be stored at –20°C.
Unit Definition: (forward reaction) One unit of hypoxanthine-guanine phosphoribosyl transferase converts 1 µmole of hypoxanthine to IMP per minute at pH 7.5 at 37°C, as measured by a coupled bacterial IMPDH (NovoCIB) enzyme system. Specific Activity: ≥ 0.8 unit/mg protein. Purity: controlled by 10%AA SDS-PAGE. |
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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. | ||
| Assay conditions: Enzymatic activity of HGPRT is measured by spectrophotometric assays in a coupled xanthine dehydrogenase (XDH, NovoCIB) system. Assays were carried out at 37°C, in 50mM Tris-HCl pH 7.5; 12mM Mg Acetate, 2mM NAD, 1mM PPi. Reaction started by adding IMP at various concentrations. NADH formation was measured in an iEMS Reader MF (Labsystems, Finland) microtiter plate reader at 340nm.
Lineweaver-Burk plotting gave a KM = 5.45µM, which is in accordance with published data (5.4µM)(1) V mouse over to enlarge |
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Download this document "NovoCIB's Human Recombinant HGPRT" 
NOVOCIB provides an HPRT Assay Kit which is coupled with an IMPDH enzyme system, rendering very easy and simple the measurement of HGPRT activities by spectrophotometry (NADH formation). This Assay Kit contains:
• Buffer (x10) and reagents for the foward reaction (IMP formation)
• Bacterial IMPDH (lyophilized) to allow a coupled enzymatic system for the spectrophotometrical measurement of IMP formed by HGPRT.
Recombinant HGPRT can be provided for positive control.
• Buffer (x10) and reagents for the foward reaction (IMP formation)
• Bacterial IMPDH (lyophilized) to allow a coupled enzymatic system for the spectrophotometrical measurement of IMP formed by HGPRT.
Recombinant HGPRT can be provided for positive control.
Download this document "NovoCIB's HGPRT Activity Assay Kit"