Diadenosine 5′,5‴-P1,P4-tetraphosphate hydrolase is present in human erythrocytes, leukocytes and platelets

doi:10.1016/1357-2725(94)00076-N

The International Journal of Biochemistry & Cell Biology

Volume 27, Issue 2, February 1995, Pages 201-206

https://doi.org/10.1016/1357-2725(94)00076-N Get rights and content

Abstract

An asymmetrically-cleaving diadenosine 5′,5‴-P¹,P⁴-tetraphosphate hydrolase (Ap₄A → ATP + AMP) is present in all higher eukaryotes and contributes to the regulation of the intracellular level of the alarmone nucleotide diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A). This enzyme has previously been isolated from unfractionated human blood cells. The aim of this report is to determine the contribution made by different blood cell types as part of our study of the roles of Ap₄A as an intra- and extracellular signalling molecule. Ap₄A hydrolase was partially purified from isolated human erythrocytes, leukocytes and platelets by high performance gel permeation chromatography and characterized by kinetic analysis and by probing immunoblots with an antibody raised against the human placental enzyme. Ap₄A hydrolase was clearly present in all three cell types. Each enzyme comprised a single polypeptide of M_r 19,200. The erythrocyte and platelet enzymes had a K_m for Ap₄A of 0.70 ± 0.05 μM (n = 3) while the K_m for the leukocyte enzyme was 1.50 ± 0.20 μM (n = 3). All three enzymes showed substrate inhibition above 10 μM Ap₄A. The specific activity of the enzyme in erythrocytes was 0.067 U/10⁶ cells, 15-fold lower than that in leukocytes and platelets. However, the erythrocyte hydrolase accounted for 97% of the total activity of unfractionated blood cells (336 U out of 346 U/ml blood). The study shows that leukocytes, platelets and erythrocytes all contain Ap₄A hydrolase activity. The last observation is of particular interest given the reported absence of Ap₄A from enucleated erythrocytes. Two possible interpretations are that the enzyme is retained from the reticulocyte stage, or that erythrocytes retain a capacity, previously unobserved, for dinucleoside polyphosphate synthesis.

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Enzymes cleaving dinucleoside polyphosphates

Cited by (15)

Biochemical and immunochemical characterisation of human diadenosine triphosphatase provides evidence for its identification with the tumour suppressor Fhit protein
2006, Biochimie
Citation Excerpt :
Concentrates of human platelets were used as a source of both platelets and leukocytes. Procedures for handling of blood cells and preparation of soluble protein extracts have been previously described in [25,29]. A small 5 ml sample of each crude homogenate was subjected to ultracentrifugation (100,000 × g, 45 min) to obtain soluble protein extracts from platelets and leukocytes.
We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit protein, a homodimer composed of ≈17 kDa monomers. It catalyses the Mg²⁺-dependent hydrolysis of diadenosine triphosphate, Ap₃A, to AMP + ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, ε-(Ap₃A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively. Human Ap₃Aase presents a native molecular mass of ≈32 kDa and no significant differences were found in K_m values (2 μM), activating effects by Mg²⁺, Ca²⁺, and Mn²⁺, optimum pH (7.0–7.2) or inhibition by Zn²⁺ and diethyl pyrocarbonate between the human enzyme and the recombinant Fhit protein. Suramin is a very potent competitive inhibitor of both human Ap₃Aase and Fhit protein with K_i values in the range 20–30 nM. Both human and rat Ap₃Aase activity co-purifies with Fhit immunoreactivity under gel filtration, ion-exchange and affinity chromatography. Homogeneous human Ap₃Aase preparations analysed by SDS-PAGE and Western blot analysis with Fhit antibodies elicit immunochemical responses corresponding to a ≈17 kDa polypeptide, indicating a dimeric structure for the enzyme Ap₃Aase. The strong inhibition of Fhit enzyme by the drug suramin, supports the need to investigate the therapeutic potential of Fhit–Ap₃Aase mediated by its interaction with suramin or related drugs.
Cloning, characterisation and crystallisation of a diadenosine 5′,5‴-P<sup>1</sup>,P<sup>4</sup>-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans
2001, Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Asymmetrically cleaving diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap₄A hydrolase. It hydrolyses Ap₄A with a K_m of 7 μM and k_cat of 27 s⁻¹ producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K_i=25 μM) and competitively by adenosine 5′-tetraphosphate with Ap₄A as substrate (K_i=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 Å and belong to either space group C222 or C222₁. Phylogenetic analysis of known and putative Ap₄A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap₄A hydrolase will help determine the basis of this grouping.
Specific and nonspecific enzymes involved in the catabolism of mononucleoside and dinucleoside polyphosphates
2000, Pharmacology and Therapeutics
This review concerns enzymes that can degrade nucleoside 5′-tetra- and pentaphosphates (p₄N and p₅N) and those that can degrade various dinucleoside polyphosphates (Np_3–6N′). Most of these enzymes are hydrolases, and they occur in all types of organisms. Certain fungi and protozoa also possess specific Np_nN′ phosphorylases. Specific p₄N hydrolases have been demonstrated in mammals and in plants. In yeast, p₄N and p₅N are hydrolyzed by exopolyphosphatases. Among other hydrolases that can degrade these minor mononucleotides are phosphatases, apyrase, and (asymmetrical) Np₄N′ hydrolase, as well as the nonspecific adenylate deaminase. Np_nN′s are good substrates for Type I phosphodiesterases and nucleotide pyrophosphatases, and diadenosine polyphosphates are easily deaminated to diinosine polyphosphates by nonspecific adenylate deaminases. Specific Np₃N′ hydrolases occur in both prokaryotes and eukaryotes. Interestingly, the human fragile histidine triad (Fhit) tumor suppressor protein appears to be a typical Np₃N′ hydrolase. Among the specific Np₄N′ hydrolases are asymmetrically cleaving ones, which are typical of higher eukaryotes, and symmetrically cleaving enzymes found in Physarum polycephalum and in many bacteria. An enzyme that hydrolyzes both diadenosine tetraphosphate and diadenosine triphosphate has been found in the fission yeast Schizosaccharomyces pombe. Its amino acid sequence is similar to that of the human Fhit/Np₃N′ hydrolase. Very recently, a typical (asymmetrical) Np₄N′ hydrolase has been demonstrated for the first time in a bacterium—the pathogenic Bartonella bacilliformis. Another novelty is the discovery of diadenosine 5′,5′′′-P¹,P ⁶-hexaphosphate hydrolases in budding and fission yeasts and in mammalian cells. These enzymes and the (asymmetrical) Np₄N′ hydrolases have the amino acid motif typical of the MutT (or Nudix hydrolase) family. In contrast, the Schizosaccharomyces pombe Ap₄A/Ap₃A hydrolase, the human Fhit protein, and the yeast Np_nN′ phosphorylases belong to a superfamily GAFH, which includes the histidine triad proteins.
Molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate hydrolases and phosphorylases
1997, International Journal of Biochemistry and Cell Biology
Diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A) pyrophosphohydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5′,5‴-P¹,P⁴-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap₄A hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap₄A hydrolase (K_m = 0.8 μM), being sensitive to inhibition by fluoride (K_i = 24 μM) and adenosine 5′-tetraphosphate (K_i = 10 nM) and yielding ATP and AMP as products. A low K_m Ap₄A hydrolase (K_m = 0.3 μM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal hydrolase, which has much higher values of K_m for Ap₄A and K_i for adenosine 5′-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequence comparisons among eukaryotic diadenosine polyphosphate hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.
The effects of diadenosine polyphosphates on the cardiovascular system
1999, Cardiovascular Research
Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation
2013, PLoS ONE

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The International Journal of Biochemistry & Cell Biology

Abstract

References (22)

Alteration of hemoglobin function by diadenosine 5′,5‴-P¹,P⁴-tetraphosphate and other alarmones

J. biol. Chem.

Human placental (asymmetrical) diadenosine 5′,5‴-P¹,P⁴-tetraphosphate hydrolase: purification to homogeneity and some properties

Protein Expr. Purif.

The presence of diadenosine 5′,5‴-P¹,P³-triphosphate (Ap₃A) in human platelets

Biochem. biophys. Res. Commun.

Diadenosine tetraphosphatase from human leukemia cells

Dinucleoside tetraphosphatase in rat liver and Artemia

Biochim. Biophys. Acta

Bis-(5′-guanosyl)tetraphosphatases in rat tissues

Biochem. J.

Nonadenylated bis(5′-nucleosidyl)tetraphosphates occur in Saccharomyces cerevisiae and Escherichia coli and accumulate upon temperature shift or exposure to cadmium

J. biol. Chem.

Abundant amounts of diadenosine 5′,5‴-P¹,P⁴-tetraphosphate are present and releasable, but metabolically inactive, in human platelets

Biochem. J.

Efficient production of chicken egg-yolk antibodies against a conserved mammalian protein

FASEB J.

Mechanism of synthesis of adenosine (5′)tetraphospho(5′)adenosine (AppppA) by aminoacyl-tRNA synthetases

Eur. J. Biochem.

Enzymes cleaving dinucleoside polyphosphates

Cited by (15)

Biochemical and immunochemical characterisation of human diadenosine triphosphatase provides evidence for its identification with the tumour suppressor Fhit protein

Cloning, characterisation and crystallisation of a diadenosine 5′,5‴-P<sup>1</sup>,P<sup>4</sup>-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans

Specific and nonspecific enzymes involved in the catabolism of mononucleoside and dinucleoside polyphosphates

Molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate hydrolases and phosphorylases

The effects of diadenosine polyphosphates on the cardiovascular system

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

General paperDiadenosine 5′,5‴-P1,P4-tetraphosphate hydrolase is present in human erythrocytes, leukocytes and platelets

Abstract

J. biol. Chem.

Protein Expr. Purif.

Biochem. biophys. Res. Commun.

Biochim. Biophys. Acta

Bis-(5′-guanosyl)tetraphosphatases in rat tissues

Biochem. J.

Nonadenylated bis(5′-nucleosidyl)tetraphosphates occur in Saccharomyces cerevisiae and Escherichia coli and accumulate upon temperature shift or exposure to cadmium

J. biol. Chem.

Abundant amounts of diadenosine 5′,5‴-P1,P4-tetraphosphate are present and releasable, but metabolically inactive, in human platelets

Biochem. J.

Efficient production of chicken egg-yolk antibodies against a conserved mammalian protein

FASEB J.

Mechanism of synthesis of adenosine (5′)tetraphospho(5′)adenosine (AppppA) by aminoacyl-tRNA synthetases

Eur. J. Biochem.

Enzymes cleaving dinucleoside polyphosphates

General paper
Diadenosine 5′,5‴-P¹,P⁴-tetraphosphate hydrolase is present in human erythrocytes, leukocytes and platelets

Abundant amounts of diadenosine 5′,5‴-P¹,P⁴-tetraphosphate are present and releasable, but metabolically inactive, in human platelets