Inhibition of IMP-specific cytosolic 5′-nucleotidase and adenosine formation in rat polymorphonuclear leucocytes by 5′-deoxy-5′-isobutylthio derivatives of adenosine and inosine

Składanowski, Andrzej C.; Sala, G; Newby, Andrew C.

doi:10.1042/bj2620203

Cited by 22 publications

(16 citation statements)

References 22 publications

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“…The enzyme is inhibited by P1 but unaffected by adenosine 5'-[a,,fmethylene]diphosphate. It shows apparent inhibition by inosine and 2',3'-dideoxyinosine, which results from the exchange of the phosphate moiety between the nucleoside and IMP (Worku & Newby, 1982;Keller et al, 1985;Johnson & Fridland, 1989;Skladanowski et al, 1989;Tozzi et al, 1991). Glycerate 2,3bisphosphate (Bontemps et al, 1988(Bontemps et al, , 1989Itoh & Yamada, 1990;Tozzi et al, 199 1) and also the diadenosine polyphosphates Ap3A, Ap4A and Ap5A are potent stimulators (Pinto et al, 1986;Itoh & Yamada, 1990).…”

Section: Soluble Formsmentioning

confidence: 99%

5′-Nucleotidase: molecular structure and functional aspects

Zimmermann

1992

Biochemical Journal

798

701

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Section: Soluble Formsmentioning

confidence: 99%

5′-Nucleotidase: molecular structure and functional aspects

Zimmermann

1992

Biochemical Journal

798

701

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“…These data suggest a selective role for cN-II in catabolism of IMP and cast doubt on whether it is active during ATP catabolism. On the other hand, the K m for AMP of cN-II measured in the presence of optimal activator, ATP, is similar to that of cN-I (approximately 5 mM) (19), and studies with selective inhibitors have also implicated cN-II in adenosine formation (20) during ATP breakdown.…”

mentioning

confidence: 96%

Distinct Roles for Recombinant Cytosolic 5′-Nucleotidase-I and -II in AMP and IMP Catabolism in COS-7 and H9c2 Rat Myoblast Cell Lines

Sala-Newby¹,

Freeman²,

Składanowski³

et al. 2000

Journal of Biological Chemistry

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Catabolism of AMP during ATP breakdown produces adenosine, which restores energy balance. Catabolism of IMP may be a key step regulating purine nucleotide pools. Two, cloned cytosolic 5-nucleotidases (cN-I and cN-II) have been implicated in AMP and IMP breakdown. To evaluate their roles directly, we expressed recombinant pigeon cN-I or human cN-II at similar activities in COS-7 or H9c2 cells. During rapid (more than 90% in 10 min) or slower (30 -40% in 10 min) ATP catabolism, cN-I-transfected COS-7 and H9c2 cells produced significantly more adenosine than cN-II-transfected cells, which were similar to control-transfected cells. Inosine and hypoxanthine concentrations increased only during slower ATP catabolism. In COS-7 cells, 5-nucleotidase activity was not rate-limiting for inosine and hypoxanthine production, which was therefore unaffected by cN-II-and actually reduced by cN-I-overexpression. In H9c2 cells, in which 5-nucleotidase activity was rate-limiting, only cN-II overexpression accelerated inosine and hypoxanthine formation. Guanosine formation from GMP was also increased by cN-II. Our results imply distinct roles for cN-I and cN-II. Under the conditions tested in these cells, only cN-I plays a significant role in AMP breakdown to adenosine, whereas only cN-II breaks down IMP to inosine and GMP to guanosine.ATP levels are a key determinant of cellular function. Formation of adenosine from AMP represents an important regulatory mechanism in cellular ATP homeostasis. In the heart, for example, adenosine causes coronary vasodilatation (1), inhibits sinoatrial and atrioventricular conduction (2), antagonises production (3) and action of catecholamines (4), causes pain (5), and contributes to ischemic preconditioning (6). We proposed that adenosine acts as a "retaliatory metabolite," being formed when there is an excess of ATP breakdown over its formation and mediating the spectrum of actions designed to restore energy balance (7,8).The pathways of ATP catabolism and adenosine formation are described in Fig. 1. The nature of the enzyme responsible for cytosolic AMP catabolism to adenosine has been the subject of much controversy. Two forms of cytosolic 5Ј-nucleotidase (cN-I and cN-II) 1 (EC 3.1.3.5) have been implicated. The cN-I was very recently cloned and shown to catalyze adenosine formation during ATP breakdown (9). The role of cN-II is less clear. Levels of expression of cN-II are usually higher in cultured cells compared with the corresponding tissue. It has therefore been suggested that cN-II is particularly important in cells with high DNA synthesis or turnover (10). The cN-II was cloned from chicken, human, and ox (11, 12) and was later shown to have activity in vitro (12, 13). Stable cell lines overexpressing cN-II were recently shown to have slightly reduced total nucleoside triphosphate pools (14). However, the function of cN-II in cells undergoing ATP breakdown has remained untested.From enzyme kinetic data, the cN-I displays a preference for AMP over IMP and is stimulated by ADP but not ATP (1...

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“…14) and more recently by a strategy that exploits differences in optimal conditions for the ubiquitous nucleotidases (17). Differential assays can take advantage of inhibitors of individual nucleotidases (8,(17)(18)(19)(20). The most active inhibitors described so far are pyrimidine nucleotide and nucleoside analogs inhibiting cN-I at nanomolar or low micromolar concentrations with up to 1000-fold selectivity for cN-I relative to cN-II or eN (18).…”

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confidence: 99%

Mammalian 5′-Nucleotidases

Bianchi

Spychała

2003

Journal of Biological Chemistry

192

184

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Nucleoside monophosphate phosphohydrolases or 5Ј-nucleotidases (members of EC 3.1.3.5 and EC 3.1.3.6) dephosphorylate non-cyclic nucleoside monophosphates to nucleosides and inorganic phosphate. Seven human 5Ј-nucleotidases with different subcellular localization have been cloned (Table I). Sequence comparisons show high homology only between cytosolic 5Ј-nucleotidase IA (cN-IA) 1 and B and between cytosolic 5Ј(3Ј)-deoxynucleotidase (cdN) and mitochondrial 5Ј(3Ј)-deoxynucleotidase (mdN). However, the existence of common motifs suggests a common catalytic mechanism for all intracellular 5Ј-nucleotidases. Some 5Ј-nucleotidases are ubiquitous (ecto-5Ј-nucleotidase (eN), cN-II, cdN, and mdN); others display tissue-specific expression (cN-I and cN-III).Here we summarize recent advances on the structure and cellular functions of the cloned 5Ј-nucleotidases. We also propose a revised nomenclature, agreed upon with other colleagues active in the nucleotidase field. Catalytic MechanismCrystal structures are known for human mdN (10) and cdN 2 and for Escherichia coli periplasmic 5Ј-nucleotidase (11), a homologue of eN. All intracellular nucleotidases share a DX-DX(V/T) motif critical for catalysis and show structural similarity to the haloacid dehalogenase superfamily of enzymes (10). eN belongs to a separate family that includes also 2Ј,3Ј-cyclic phosphodiesterases and apyrases (11).The crystal structure of mdN and work on the active site of cN-II form the basis for a reaction mechanism of intracellular 5Ј-nucleotidases (10, 12). The reaction creates a phosphoenzyme intermediate involving the first aspartate in the DX-DX(V/T) motif (12). A detailed scheme of the catalytic process derived from the crystal structure of mdN (10) involved both aspartates in the above motif (Fig. 1). The first (Asp-41) generates a pentacovalent phosphorus intermediate with similar basic organization as the intermediate detected in the structure of ␤-phosphoglucomutase (13). The x-ray structure of cdN suggests a catalytic mechanism identical with that of mdN. Differences within the active sites account for differences in substrate specificity (10).2 Using the structurally important residues the best alignment was between the two deoxynucleotidases and cN-III (10). Two 5Ј-nucleotidases, cN-II and cN-III, exhibit phosphotransferase activity (for reviews see Refs. 14 and 15) possibly because of higher stability of the phosphoenzyme intermediate or faster exchange of the nucleoside product with the nucleoside acceptor. The active site of E. coli 5Ј-nucleotidase, the paradigm for eN, contains two zinc ions and the catalytic dyad . No phosphoenzyme intermediate is formed during catalysis, but a water molecule performs the nucleophilic attack on the phosphate (16). Properties, Detection, and Inhibition of 5-NucleotidasesAll 5Ј-nucleotidases have relatively broad substrate specificities. In agreement with the structural information on the active sites (10, 11), all family members except eN are absolutely dependent on magnesium for activity. Table II s...

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Inhibition of IMP-specific cytosolic 5′-nucleotidase and adenosine formation in rat polymorphonuclear leucocytes by 5′-deoxy-5′-isobutylthio derivatives of adenosine and inosine

Cited by 22 publications

References 22 publications

5′-Nucleotidase: molecular structure and functional aspects

5′-Nucleotidase: molecular structure and functional aspects

Distinct Roles for Recombinant Cytosolic 5′-Nucleotidase-I and -II in AMP and IMP Catabolism in COS-7 and H9c2 Rat Myoblast Cell Lines

Mammalian 5′-Nucleotidases

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