NOVOCIB's human cytosolic IMP/GMP specific 5'-nucleotidase/phosphotransferase II (cN-II) is a pure and active protein of 65kDa cloned by RT-PCR amplification of mRNA extracted from human hepatoma cells and expressed in E.coli. The sequence of the cloned NT5C2 gene (GenBank accession number P49902) was confirmed by DNA sequencing (100% identity).
Cytosolic 5'-nucleotidase II is one of the seven known mammalian nucleotidases⁽¹⁾ that specifically catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5�-monophosphates (IMP, dIMP, GMP, dGMP) and regulates cellular pool of IMP and GMP^(2,3). The enzyme also acts as a phosphotransferase catalyzing the transfer of a phosphate from nucleoside monophosphate to a nucleoside acceptor � preferentially inosine and deoxyinosine. Unlike the other 5�-nucleotidases, cN-II is allosterically regulated by adenine/guanine nucleotides and 2,3-biphosphoglycerate⁽⁴⁾.
In addition, cytosolic 5'-nucleotidase II phosphorylates anti-viral and anti-tumour nucleoside analogues such as 2�3�-dideoxyinosine, carbovir⁽⁵⁾, acyclovir⁽⁶⁾ and ribavirin⁽⁷⁾.

Assay condition: Phosphotransferase activity of human recombinant cN-II was followed at 37°C, in 100mM Tris-HCl pH 9; 100mM KCl, 20mM MgCl2, 0.5mM KH2PO4, 5mM DTT, 5mM NAD, 50mU/mL IMPDH (NovoCIB). 5mM GMP was used as phosphate donor and 5mM inosine as - phosphate acceptor. The formation of IMP from inosine was followed at 340nm through coupling to recombinant IMP-dehydrogenase (IMPDH, NovoCIB). In the presence of NAD, IMPDH catalyzes irreversible conversion of IMP to XMP with concomitant reduction of NAD to NADH2 (specific absorbance at 340nm).

Assay condition: 5�-nucleotidase activity of cN-II is followed in an irreversible spectrophotometric assay using coupled purine nucleoside phosphorylase - xanthine dehydrogenase system (2,5 mU/ml each). Assays are carried out at 37�C, at 50mM Tris-HCl pH7,6; 100mM KCl, 20mM MgCl2, 500�M KH2PO4, 5mM DTT, 119 nM cN-II and various concentrations of IMP. Reaction is followed at 295 nm. IMP is purchased from MP Biochemicals. PNP and XDH are produced and purified by NovoCIB.

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References (with links to PubMed) 1. V. Bianchi and J. Spychala (2003): Mammalian 5'-nucleotidases J. Biol. Chem. 278(47), 46195-8 2. S. Allegrini et al. (1997): Bovine cytosolic IMP/GMP-specific 5'-nucleotidase: cloning and expression of active enzyme in Escherichia coli Biochem. J. 328(Pt2), 483-487 3. P. L. Ipata and M. G. Tozzi (2006): Recent advances in structure and function of cytosolic IMP-GMP specific 5'-nucleotidase II (cN-II) Purinergic Signal. 2(4), 669-675 4. J. Spychala et al. (1988): High Km Soluble 5�-nucleotidase from human placenta J. Biol. Chem. 263(35):18759-65 5. M. A. Johnson and A. Fridland (1989): Phosphorylation of 2',3'-dideoxyinosine by cytosolic 5'-nucleotidase of human lymphoid cells Mol. Pharmacol. 36(2), 291-295 6. P. M. Keller (1985): Cytoplasmic 5'-nucleotidase catalyzes acyclovir phosphorylation J. Biol. Chem. 260(15), 8664-7 7. J. Z. Wu et al. (2005): Phosphorylation of ribavirin and viramidine by adenosine kinase and cytosolic 5'-nucleotidase II: Implications for ribavirin metabolism in erythrocytes Antimicrob. Agents Chemother. 49(6): 2164-71