Bovine cytosolic IMP/GMP-specific 5′-nucleotidase: cloning and expression of active enzyme in Escherichia coli

Abstract

A cDNA coding for bovine cytosolic IMP/GMP-specific 5'-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5' and 3' rapid amplification of cDNA ends protocols (5' and 3' RACE). Two products were isolated: a 5' RACE 1.6 kb fragment and a 3' RACE 2.0 kb fragment, with an overlapping region of 505 bp, leading to a total length of approx. 2951 bp. The similarity in the coding region to that of the human 5'-nucleotidase cDNA sequence [Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem. Biophys. Res. Commun. 205, 917-922], indirectly identified as a 5'-nucleotidase, was 94% and the deduced amino acid sequences were 99.5% identical. The bovine cDNA sequence included the sequences codifying for six peptides obtained from 5'-nucleotidase/phosphotransferase purified from calf thymus. Northern blots of human mRNA species from different tissues showed a 3.6 kb mRNA expressed at equal levels in most tissues. The cDNA was cloned into a pET-28c expression vector and the protein obtained after induction had a molecular mass of 61 kDa under SDS/PAGE. It exhibited both 5'-nucleotidase and phosphotransferase activity, as well as immunological and kinetic properties similar to those of the enzyme purified from calf thymus. This is the first time that a fully active recombinant 5'nucleotidase has been described.

Suppression of 5′-Nucleotidase Enzymes Promotes AMP-activated Protein Kinase (AMPK) Phosphorylation and Metabolism in Human and Mouse Skeletal Muscle

“…Because NT5C1A is expressed at low levels in cultured human myotubes, we silenced NT5C2, an IMP-preferring enzyme that has overlapping specificity for AMP (18,19). We hypothesized that siRNA against NT5C2 may increase the availability of intracellular AMP and IMP, which would alter the AMP/ATP ratio and increase AMPK activity.…”

“…5Ј-Nucleotidases have broad substrate specificity and are involved in maintaining cellular nucleotide and nucleoside levels by catalyzing the hydrolysis of either AMP to adenosine and phosphate or IMP to inosine and phosphate (15). Tissue expression profiling studies have revealed that NT5C1A and NT5C2 are expressed in skeletal muscle (17)(18)(19). NT5C1A has a higher affinity for AMP, whereas NT5C2 has a higher affinity for IMP, and both are relevant for the intercellular turnover of AMP in skeletal muscle (20).…”

Expression of high Km 5′-nucleotidase in leukemic blasts is an independent prognostic factor in adults with acute myeloid leukemia

“…18,32,33 The high Km 5NT (also designated as cN-II; accession No. D38524) studied in this patient population 32,[34][35][36] was the first cytoplasmic 5NT cloned. 33 5Ј-3Ј-Nucleotidase (dNT-1; also described as PN-II 37 ; accession No.…”

Crystal Structure of Human Cytosolic 5′-Nucleotidase II

“…The cN-II was first purified from chicken liver (10) and has been intensively investigated in vertebrates (11). As substrates it prefers the 6-hydroxypurine nucleoside monophosphates IMP, dIMP, GMP, dGMP, and XMP (10,(12)(13)(14)(15) and functions as a tetramer (12, 16 -18). It is ubiquitously expressed and is likely to play an important role in the regulation of purine nucleotide interconversions and in the regulation of IMP and GMP pools within the cell (1).…”

“…Phosphate donors include any 6-hydroxypurine monophosphate substrate of the nucleotidase reaction (13)(14)(15)(22)(23)(24).…”