A new nucleotidase of rat liver with activity toward 3′- and 5′-nucleotides

Fritzson, Per; Smith, Inger

doi:10.1016/0005-2744(71)90040-4

Cited by 52 publications

(21 citation statements)

References 34 publications

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“…7). The large effect on deoxyuridine agrees with the preference of the enzyme for dUMP as substrate (14,15). In 293 cells induction of dNT-1 also increased thymidine excretion.…”

Section: Discussionsupporting

confidence: 68%

Cytosolic High K 5′-Nucleotidase and 5′(3′)-Deoxyribonucleotidase in Substrate Cycles Involved in Nucleotide Metabolism

Gazziola

Ferraro

Moras

et al. 2001

Journal of Biological Chemistry

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5-Nucleotidases are the catabolic members of the substrate cycles postulated to be involved in the regulation of intracellular deoxyribonucleoside triphosphate pools. Here, we attempt to identify the nature of the nucleotidases. Earlier, we constructed various mammalian cell lines that can be induced to overproduce the high K m 5-nucleotidase (hkm-NT) or the 5(3)-deoxynucleotidase (dNT-1). Now we labeled control and induced human 293 cells and hamster V79 cells with radioactive hypoxanthine or uridine and during a chase measured quantitatively the metabolism of ribo-and deoxyribonucleotides, DNA replication, and excretion of nucleosides into the medium. Overproduction of hkm-NT greatly increased excretion of inosine and guanosine but did not affect adenosine or deoxyribonucleosides. dNT-1 overproduction increased excretion of deoxycytidine, thymidine, and in particular deoxyuridine but also uridine and cytidine. We conclude that the hkm-NT is not involved in the regulation of deoxyribonucleotide pools but affects IMP and GTP pools. dNT-1, instead, appears to be the catabolic arm of substrate cycles regulating pyrimidine nucleotide pools.DNA replication and repair requires a continuous and balanced supply of the four deoxyribonucleoside triphosphates (dNTPs).1 Precursor pools are very small, suffice only for a few minutes of DNA replication in mammalian cells, and have to be continuously replenished during S phase (1). Imbalances in pool sizes are genotoxic and may in severe cases lead to cell death (reviewed in Ref. 2). A network of biosynthetic and catabolic enzymes regulates the size of each pool with the enzyme ribonucleotide reductase playing a pivotal role (1). This enzyme not only directs the total flow of metabolites into DNA but also, via an exquisite allosteric mechanism, divides the flow into separate channels to maintain the balance between the four pools.A second level of control comes from the dynamic interplay between kinases, catalyzing the salvage of deoxyribonucleosides, and nucleotidases, involved in the catabolism of deoxyribonucleotides (1). The two groups of enzymes form substrate (or futile) cycles (3, 4) by catalyzing two opposite irreversible reactions whose net result is the hydrolysis of ATP to ADP and P i (Fig. 1). This seemingly wasteful exercise provides the cell with a mechanism for the fine tuning of dNTP pools (1) that relies on the difference in cell permeability between nucleosides and nucleotides. Nucleosides move freely between the inside and outside of the cell, whereas nucleotides remain trapped inside (Fig. 1). When catabolism outweighs anabolism, deoxyribonucleosides accumulate and are excreted. In the opposite situation, they are imported and further metabolized to dNTPs. The excretion or the import of a nucleoside over the cell membrane can be used as a measure of the activity of substrate cycles."Auxiliary" catabolic enzymes (deaminases, phosphorylases, and hydrolases) remove some of the nucleosides and thereby shift the equilibrium in the direction of catabolism (Fig. ...

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“…7). The large effect on deoxyuridine agrees with the preference of the enzyme for dUMP as substrate (14,15). In 293 cells induction of dNT-1 also increased thymidine excretion.…”

Section: Discussionsupporting

confidence: 68%

Cytosolic High K 5′-Nucleotidase and 5′(3′)-Deoxyribonucleotidase in Substrate Cycles Involved in Nucleotide Metabolism

Gazziola

Ferraro

Moras

et al. 2001

Journal of Biological Chemistry

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“…121 The deoxyribonucleotidase family includes one of the major types of 5′ (3′)-deoxyribonucleotidases responsible for dephosphorylating uracil and thymine deoxyribonucleotides. [122][123][124] The eukaryotic forms do not group together in phylogenetic analysis, suggesting that they might have been acquired from bacterial or phage sources on multiple occasions. The presence in large DNA viruses and mitochondria is consistent with other similarities between their DNA replication processes 125,126 and is indicative of the similar selective pressures faced by these replicons from excess uracil and thymine dNTs.…”

Section: Simple Bi-helical Cap Familiesmentioning

confidence: 99%

Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes

Burroughs¹,

Allen²,

Dunaway‐Mariano³

et al. 2006

Journal of Molecular Biology

387

520

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The HAD (haloacid dehalogenase) superfamily includes phosphoesterases, ATPases, phosphonatases, dehalogenases, and sugar phosphomutases acting on a remarkably diverse set of substrates. The availability of numerous crystal structures of representatives belonging to diverse branches of the HAD superfamily provides us with a unique opportunity to reconstruct their evolutionary history and uncover the principal determinants that led to their diversification of structure and function. To this end we present a comprehensive analysis of the HAD superfamily that identifies their unique structural features and provides a detailed classification of the entire superfamily. We show that at the highest level the HAD superfamily is unified with several other superfamilies, namely the DHH, receiver (CheY-like), von Willebrand A, TOPRIM, classical histone deacetylases and PIN/FLAP nuclease domains, all of which contain a specific form of the Rossmannoid fold. These Rossmannoid folds are distinguished from others by the presence of equivalently placed acidic catalytic residues, including one at the end of the first core β-strand of the central sheet. The HAD domain is distinguished from these related Rossmannoid folds by two key structural signatures, a "squiggle" (a single helical turn) and a "flap" (a beta hairpin motif) located immediately downstream of the first β-strand of their core Rossmanoid fold. The squiggle and the flap motifs are predicted to provide the necessary mobility to these enzymes for them to alternate between the "open" and "closed" conformations. In addition, most members of the HAD superfamily contains inserts, termed caps, occurring at either of two positions in the core Rossmannoid fold. We show that the cap modules have been independently inserted into these two stereotypic positions on multiple occasions in evolution and display extensive evolutionary diversification independent of the core catalytic domain. The first group of caps, the C1 caps, is directly inserted into the flap motif and regulates access of reactants to the active site. The second group, the C2 caps, forms a roof over the active site, and access to their internal cavities might be in part regulated by the movement of the flap. The diversification of the cap module was a major factor in the exploration of a vast substrate space in the course of the evolution of this superfamily. We show that the HAD superfamily contains 33 major families distributed across the three superkingdoms of life. Analysis of the phyletic patterns suggests that at least five distinct HAD proteins are traceable to the last universal common ancestor (LUCA) of all extant organisms. While these prototypes diverged prior to the emergence Abbreviations used: HAD, haloacid dehalogenase; PNKP, polynucleotide kinase phosphatase; KDO 8-P, deoxy-Dmannose-octulosonate 8-phosphate; CTD, carboxyl-terminal domain; PNKP, polynucleotide kinase phosphatase; LUCA, last universal common ancestor.E-mail address of the corresponding author: aravind@ncbi.nlm.nih.gov of the LUCA, t...

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“…Gel-permeation chromatography (Table 2) indicated that about 15% of the crude supernatant-fraction activity towards UMP was due to enzyme(s) different from the alkaline 5'-nucleotidase. Such enzymes could be cytosolic 5'-nucleotidase and the deoxyribonucleoside-activated nucleotidase, with pH optima 6.3 and 6.0 respectively, which may both exert some activity also at pH 8.1 (Fritzson, 1969;Fritzson & Smith, 1971). Contribution of these enzymes to the supernatant-fraction activity could explain a slightly smaller decrease in UMP and IMP dephosphorylation in regenerating liver compared with AMP dephosphorylation, since both enzymes show increased activity in regenerating liver (Fritzson, 1978;Tjernshaugen & Fritzson, 1984) and exhibit very low activity towards AMP at the substrate concentrations used.…”

Section: Discussionmentioning

confidence: 99%

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5′-nucleotidase

Fritzson¹,

Haugen²,

Tjernshaugen³

1986

Biochemical Journal

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An alkaline 5′-nucleotidase with properties similar to those of membrane-bound 5′-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.

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A new nucleotidase of rat liver with activity toward 3′- and 5′-nucleotides

Cited by 52 publications

References 34 publications

Cytosolic High K 5′-Nucleotidase and 5′(3′)-Deoxyribonucleotidase in Substrate Cycles Involved in Nucleotide Metabolism

Cytosolic High K 5′-Nucleotidase and 5′(3′)-Deoxyribonucleotidase in Substrate Cycles Involved in Nucleotide Metabolism

Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5′-nucleotidase

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