Cytosolic 5' Nucleotidase II
Various cytotoxic nucleoside analogs (NA) have been employed in the treatment of hematologic malignancies. These NA include pyrimidine analog cytosine arabinoside (ara-c) and purine analogs, cladribine and fludarabine. These NA become therapeutically effective only after phosporylation to the triphosphate level. The 5¿-nucleotidases (5¿-NTs) dephosphorylate the monophosphate form of NA and, therefore, may affect the pharmacological activity of these antimetabolites. Several 5¿-NTs attached to membranes or present in the cytosol or in mitochondri. There are two cytosolic cyclic 5’nucleotidases are cN-II and dNT-1 and one mitochondrial (dNT-2) nucleotidase known to be present in the cells. cN-I plays a significant role in AMP breakdown to adenosine whereas cN-II breaks down IMP to inosine and GMP to guanosine. The primary sequence of CNI is unrelated to CNII or ecto-5’nucleotisdase (e-N). The tissue distribution of cNI cNII is different, cNI is found only in vertebrate heart where as cNII is expressed in heart, brain and muscle. Sequence alignments of cytosolic 5¿-nucleotidase, along with other nucleotidases, suggest that cytosolic 5’nucleotidase belong to a large superfamily of hydrolases with different substrate specificities and functional roles. Overproduction of cN-II could lead to resistance against anti-cancer drugs based on purine analogs. cN-II is Mg2+-dependent, regulated and stabilized by several factors such as allosteric effectors ATP and 2,3-DPG, although these are not directly involved in the reaction stoichiometry (2).
IMP and GMP and its derivatives specific cytosolic 5¿-nucleotidase (cNII) acts through the formation of a phosphoenzyme intermediate. Phosphate either released leading to 5¿-mononucleotide hydrolysis or transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors (3). Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. There exist a putative phsohorylation site on the cNII near C-termianl end of the protein. FabGennix Int. Inc., has also produced a phospho-specific antibody to phospho-serine residue at this site (phosphorylation site: ILFRSG(Sp)RQTLf). The CNII exist as monomer (40 kDa) and multimeric forms in various tissues.
Anti-Phospho-cNII-selective antibodies were generated using kinase site (SLFRSG SR(p)QTLF, the peptide was post-synthetically modified to achieve the desired antigenicty. The affinity purified version of this antibody (PcNII-140AP) was isolated form immobilized antigen based affinity chromatography and adsorbed on non-phospho are represented as pure IgG fractions stabilized in antibody stabilization buffer. The mono-epitope specific polyclonal antibody (cNII-101AP) strongly label a 40-42 kDa cNII and its multimerric forms in PC-cNII samples. A phospho-specific antibody to cNII is also available form FabGennix Int. Inc., (cat. # cNII-140AP). The cNII antibodies can be conjugated as HRP or alkaline conjugates for IHC, Confocal, WB analyses at a nominal fee. FabGennix Inc. will also conjugate antibodies with fluorescent probes upon request at extra charge. FabGennix Inc. also provides antibodies against other diagnostic/neomarkers, the list of these antibodies can be obtained at www.FabGennix.com under Antibodies to diagnostic markers. Limited quantities of antigens are also available. Please enquire for their availability before ordering.
For research use only, not for diagnostic or therapeutic use.
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