Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. We have previously purified PN-I, a pyrimidine nucleotidase whose deficiency is associated with hemolytic anemia. Computer-aided analysis of PN-I tryptic and CNBr peptide sequences revealed substantial identity with tryptic peptide sequences reported for p36, an alpha-interferon-induced protein. PN-I partial sequences were matched through the expressed sequence tag database with different human complementary DNA (cDNA) clones, whose sequences were exploited to screen a human placenta cDNA library. PN-I cDNA, coding for a 286-residue protein, was expressed in Escherichia coli, yielding a fully active recombinant enzyme. The recombinant protein sequence comprised the peptide sequences determined for PN-I and p36. Rabbit antisera raised against two peptides deriving from p36 and PN-I tryptic digestions, respectively, recognized both wild-type and recombinant PN-I. Molecular properties of the two proteins were essentially the same. We conclude that p36 and PN-I are identical proteins. (Blood. 2000;96:1596-1598)
Two cytoplasmic forms of pyrimidine nucleotidase (PN-I and PN-II) have been purified from human erythrocytes to apparent homogeneity and partially characterized. They preferentially hydrolyse pyrimidine 5'-monophosphates and 3'-monophosphates respectively. PN-I and PN-II operate as interconverting activities, capable of transferring the phosphate from the pyrimidine nucleoside monophosphate donor(s) to various nucleoside acceptors, including important drugs like 3'-azido-3'-deoxy-thymidine (AZT), cytosine-beta-D-arabinofuranoside (AraC) and 5-fluoro-2'-deoxy-uridine (5FdUrd), pyrimidine analogues widely used in chemotherapy. Kinetic analysis showed linear behaviour for both PN-I and PN-II. PN-I phosphotransferase activity revealed higher affinity for oxynucleosides with respect to deoxy-nucleosides, whereas the contrary seems to be true for PN-II. These results show for the first time that soluble pyrimidine nucleotidases are endowed with pyrimidine-specific phosphotransferase activity.
Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. We have previously purified PN-I, a pyrimidine nucleotidase whose deficiency is associated with hemolytic anemia. Computer-aided analysis of PN-I tryptic and CNBr peptide sequences revealed substantial identity with tryptic peptide sequences reported for p36, an alpha-interferon-induced protein. PN-I partial sequences were matched through the expressed sequence tag database with different human complementary DNA (cDNA) clones, whose sequences were exploited to screen a human placenta cDNA library. PN-I cDNA, coding for a 286-residue protein, was expressed in Escherichia coli, yielding a fully active recombinant enzyme. The recombinant protein sequence comprised the peptide sequences determined for PN-I and p36. Rabbit antisera raised against two peptides deriving from p36 and PN-I tryptic digestions, respectively, recognized both wild-type and recombinant PN-I. Molecular properties of the two proteins were essentially the same. We conclude that p36 and PN-I are identical proteins. (Blood. 2000;96:1596-1598)
Br. J. Haematol. 122, 847-851 (2003)[PubMed:12930399]
Pyrimidine 5'-nucleotidase deficiency is a rare autosomal recessive disorder characterized by haemolytic anaemia, marked basophilic stippling and accumulation of pyrimidine nucleotides within the erythrocytes. The gene encoding for this enzyme (P5'N-1) has been cloned recently, and seven mutations have so far been identified in 11 unrelated families. We describe the haematological and molecular characteristics of six unrelated Italian patients affected by pyrimidine 5'-nucleotidase deficiency (one from northern and five from southern Italy). The sequence of the complete P5'N-1 gene showed the presence of four different new mutations: a missense mutation AAT-AGT at codon 190 (Asn-Ser), one splicing mutation (IVS9-1 g-c) and two frameshift mutations, DelG576 and InsGG743. Although the molecular defect was homozygous in all patients but one, parents' consanguinity could be confirmed in only one case. InsGG743 was detected in two cases, and DelG576 was found in three patients originating from southern Italy, suggesting a possible geographical distribution of the genetic defect. Haematological data showed the presence of peripheral spherocytosis in all cases, although only one had a concomitant membrane defect. An increase in serum ferritin levels was observed in the splenectomized patients, suggesting that the iron status of these subjects should be monitored and that they should be investigated for potential additional risk factors for iron accumulation.
Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. Pyrimidine 5'-nucleotidase, PN-I, has been purified and characterized. The molecular and enzymatic properties determined for the enzyme shows a 36-kDa and 5.1 pI monomeric protein with no disulfide bridges and no phosphate content. The activity is dependent on Mg(2+), while it is inactivated by heavy metals and by thiol-reactive reagents. PN-I is specific for pyrimidine nucleoside monophosphates, including the antineoplastic agents 5'-AZTMP and 5'-Ara-CMP. PN-I possess phosphotransferase activity able to exchange phosphate between pyrimidine nucleoside monophosphates and pyrimidine nucleosides, including AZT and Ara-Cyd. Amino acid sequence has been obtained from tryptic and CNBr peptides. PN-I cDNA sequence, coding for a 286-residue protein, has been retrieved from tag database, amplified by PCR, and expressed in Escherichia coli. The recombinant protein was fully active and showed identical properties with respect to PN-I. Substantial identity has been revealed with the partial sequences reported for p36, an alpha-interferon-induced protein. The significance of this identity is discussed.
Interacting selectively and non-covalently with a nucleotide, any compound consisting of a nucleoside that is esterified with (ortho)phosphate or an oligophosphate at any hydroxyl group on the ribose or deoxyribose.
The chemical reactions and pathways involving a nucleotide, a nucleoside that is esterified with (ortho)phosphate or an oligophosphate at any hydroxyl group on the glycose moiety; may be mono-, di- or triphosphate; this definition includes cyclic nucleotides (nucleoside cyclic phosphates).
The chemical reactions and pathways involving any pyrimidine nucleoside, one of a family of organic molecules consisting of a pyrimidine base covalently bonded to ribose (a ribonucleoside) or deoxyribose (a deoxyribonucleoside).
Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. We have previously purified PN-I, a pyrimidine nucleotidase whose deficiency is associated with hemolytic anemia. Computer-aided analysis of PN-I tryptic and CNBr peptide sequences revealed substantial identity with tryptic peptide sequences reported for p36, an alpha-interferon-induced protein. PN-I partial sequences were matched through the expressed sequence tag database with different human complementary DNA (cDNA) clones, whose sequences were exploited to screen a human placenta cDNA library. PN-I cDNA, coding for a 286-residue protein, was expressed in Escherichia coli, yielding a fully active recombinant enzyme. The recombinant protein sequence comprised the peptide sequences determined for PN-I and p36. Rabbit antisera raised against two peptides deriving from p36 and PN-I tryptic digestions, respectively, recognized both wild-type and recombinant PN-I. Molecular properties of the two proteins were essentially the same. We conclude that p36 and PN-I are identical proteins. (Blood. 2000;96:1596-1598)
Protein involved in the biochemical reactions of nucleotides. Nucleotides are phosphate esters of a nucleoside consisting of a purine or pyrimidine base linked to ribose or deoxyribose phosphates.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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