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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The metabolic fate of transported guanosine was examined in adult rat cardiac myocytes. Freshly isolated cells were incubated with 50 microM 8-[3H]-guanosine and the purine nucleoside phosphorylase (PNP) inhibitor acyclovir, and the nucleotide products extracted and examined for radiolabel distribution. Acyclovir inhibited guanosine incorporation into the 5'-nucleotide pool up to 66%. The drug did not inhibit guanosine transport. Other experiments using 5'-[3H]-guanosine and 8-[14C]-guanosine in concert as metabolic tracers showed both tritium and radiocarbon in the guanine nucleotide products. We concluded from this study that both a kinase (probably adenosine kinase) and the enzyme pair purine nucleoside phosphorylase/hypoxanthine-guanine phosphoribosyltransferase are responsible for guanosine salvage in heart cells.
J Mol Cell Cardiol 1992 Jul
PMID:Guanosine metabolism in adult rat cardiac myocytes: inhibition by acyclovir and analysis of a metabolic pathway. 140 8

We employed an isolated guinea-pig heart model perfused at constant pressure (70 cmH2O) to test the hypothesis that inhibition of adenosine metabolism increases interstitial adenosine concentrations (as measured with epicardial discs) and coronary flow. Iodotubercidin (ITU, 1 microM) and EHNA (erythro-9-[2-hydroxy-3-nonyl] adenine, 5 microM) were used to inhibit adenosine kinase and deaminase, respectively during control conditions and during metabolic stimulation with 1 microM isoproterenol. The adenosine receptor blocker 8-phenyltheophylline (8-PT) was used during control conditions to assess whether the response seen was adenosine specific. ITU plus EHNA decreased heart rate (202 +/- 10 to 136 +/- 11 beats/min) and increased coronary flow (8.2 +/- 0.3 to 12.4 +/- 0.9 ml/min/g) without a change in MVO2, developed pressure or dP/dt. ITU plus EHNA increased adenosine concentrations in epicardial fluid (0.24 +/- 0.07 microM to 1.02 +/- 0.09 microM) and venous effluent (40 +/- 3 nM to 262 +/- 32 nM) during control conditions, and adenosine release increased from 389 +/- 96 pmols/min/g to 3480 +/- 365 pmols/min/g. 8-PT infusion reversed the effects on heart rate and coronary flow and resulted in a persistent elevation of epicardial fluid adenosine concentrations. During metabolic stimulation with 1 microM isoproterenol, ITU plus EHNA significantly limited the increase in heart rate and ventricular developed pressure and dP/dt while coronary flow increased to a significantly greater extent. Myocardial oxygen consumption was similar during metabolic stimulation between the two groups (vehicle vs. ITU plus EHNA). Epicardial fluid adenosine concentration in the vehicle-treated group increased from 0.17 +/- 0.3 microM to 0.34 +/- 0.02 microM at 15 min of isoproterenol stimulation whereas it increased from 1.10 +/- 0.02 microM to 2.90 +/- 0.46 microM in the ITU plus EHNA-treated group. Inhibition of adenosine metabolism during metabolic stimulation significantly increased venous adenosine concentrations and adenosine release and reduced inosine and hypoxanthine release proportionately. The release of adenosine+inosine+hypoxanthine was unchanged. Inhibition of adenosine metabolism provides evidence supporting the hypothesis that adenosine plays a role in regulating coronary vascular resistance as well as influencing heart rate and ventricular inotropy.
J Mol Cell Cardiol 1992 Nov
PMID:Inhibition of adenosine metabolism increases myocardial interstitial adenosine concentrations and coronary flow. 147 23

Adenosine kinase (ATP, adenosine 5'-phosphotransferase, E.C. 2.7.1.20) from Leishmania donovani, unlike adenosine kinase from other known eukaryotic sources, does not elicit an inhibitory response at high concentrations of adenosine. The mechanistic basis for this unique catalytic behavior of the parasite enzyme has been probed with the help of chemical modification and enzyme inhibition kinetics experiments. The use of cysteine-directed reagents has shown that chemical integrity of cysteinyl residues is essential for the expression of functional activity of the enzyme. Thiol group titration revealed that the enzyme contains 3 cysteine residues. However, in contrast to adenosine kinase from other sources, inactivation of the parasite enzyme could be correlated with alkylation of 2 cysteinyl residues. Adenosine, but not ATP, protected 2 thiols against -SH blocker-mediated inactivation of the enzyme. The thiol groups were shown to map at positions corresponding to approximately 16, 22, and 36 kDa sites from the protein's N-terminal end. The functions of 2 thiols at the catalytic site were functional thiol groups yielded a 'protection constant' (KpAd) of 3.4 microM, while the dissociation constant (KsAD) of the enzyme-substrate complex was 2.7 microM, hence supporting involvement of the same in both processes, namely catalysis and protection. The overall results were therefore interpreted as showing that (a) the leishmanial enzyme, in contrast to adenosine kinase from other sources, contains 2 functional thiol groups at the catalytic site; and (b) the enzyme binds adenosine exclusively through the catalytic site and as a consequence is not amenable to inhibition at high adenosine concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Biochem Parasitol 1992 May
PMID:Active site thiol(s) in Leishmania donovani adenosine kinase: comparison with hamster enzyme and evidence for the absence of regulatory adenosine binding site. 162 5

Our earlier work on reperfusion showed that adult rat hearts released almost twice as much purine nucleosides and oxypurines as newborn hearts did [Am J Physiol 254 (1988) H1091]. A change in the ratio anabolism/catabolism of adenosine could be responsible for this effect. We therefore measured the activity of adenosine kinase, adenosine deaminase, nucleoside phosphorylase and xanthine oxidoreductase in homogenates of hearts and myocytes from neonatal and adult rats. In hearts the activity of adenosine deaminase and nucleoside phosphorylase (10-20 U/g protein) changed relatively little. However, adenosine kinase activity decreased from 1.3 to 0.6 U/g (P less than 0.025), and xanthine oxidoreductase activity increased from 0.02 to 0.85 U/g (P less than 0.005). Thus the ratio in activity of these rate-limiting enzymes for anabolism and catabolism dropped from 68 to 0.68 during cardiac development. In contrast, the ratio in myocytes remained unchanged (about 23). The large difference in adenosine anabolism/catabolism ratio, observed in heart homogenates, could explain why ATP breakdown due to hypoxia is lower in neonatal than in adult heart. Because this change is absent in myocytes, we speculate that mainly endothelial activities of adenosine kinase and xanthine oxidoreductase are responsible for this shift in purine metabolism during development.
J Mol Cell Cardiol 1990 Oct
PMID:Ischemic nucleotide breakdown increases during cardiac development due to drop in adenosine anabolism/catabolism ratio. 209 32

Carbovir (CBV) is a highly selective carbocyclic nucleoside inhibitor of HIV replication in human lymphocytes and is potentially useful in the treatment of AIDS [Vince et al. (1988) Biochem. Biophys. Res. Commun. 156, 1046-1053]. Using human lymphoid cells severely deficient in nucleoside kinases, we were able to identify the route of activation of CBV metabolism. The present studies have demonstrated that CBV is anabolized to the mono-, di-, and triphosphates and to guanosine 5'-triphosphate in CCRF-CEM cells. Conversion to GTP amounted to 15-20% of the total analogue nucleotides formed in the cells and may arise from CBV through depurination and salvage via HGPRT. Evidence was obtained that neither deoxycytidine kinase, adenosine kinase, or mitochondrial deoxyguanosine kinase is primarily involved in the initial step of phosphorylation of CBV in CCRF-CEM cells. In contrast, earlier studies [Johnson & Fridland (1989) Mol. Pharmacol. 36, 291-295] showed that a cytosolic 5'-nucleotidase catalyzes the activation of CBV to the monosphosphate. Other biochemical effects examined showed that the nucleobases hypoxanthine and adenine, but not guanine, their respective nucleosides, and the dideoxynucleosides 2',3'-dideoxyinosine, 2',3'-dideoxyguanosine, and 3'-azido-3'-deoxythymidine produced significant increased accumulation of CBV nucleotides in CEM cells. The exact mechanism for this potentiation of CBV phosphorylation has not been elucidated but may be due to a modulating effect of intracellular nucleotides on 5'-nucleotidase activity.
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PMID:Metabolism of the carbocyclic nucleoside analogue carbovir, an inhibitor of human immunodeficiency virus, in human lymphoid cells. 227 22

Neplanocin A [(-)-9-[trans-2',trans-3'-dihydroxy-4'-(hydroxymethyl)-cyclopent-4 '- enyl]-adenine] and 9-[trans-2',trans-3'-dihydroxycyclopent-4'-enyl]-adenine (1) and -3-deazaadenine (2) are potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) in mouse L929 cells. When cells were treated for 15 min with varying concentrations of the drugs, the IC95 values (concentration needed to produce 95% inhibition of AdoHcy hydrolase) for neplanocin A, 1, and 2 were determined to be 0.2 microM, 0.5 microM, and 0.5 microM, respectively. Incubation of L929 cells with 1.0 microM concentrations of neplanocin A, 1, or 2 produced rapid inactivation of AdoHcy hydrolase (within 30 min the enzyme was 95% inhibited), which persisted for at least 72 hr. At lower concentrations (0.032 microM), substantial recovery of AdoHcy hydrolase activity was noted after 48 and 72 hr in cultures treated with neplanocin A but not in cultures treated with 1 or 2. L929 cells treated with neplanocin A, 1 or 2 showed a rapid increase in intracellular levels of AdoHcy (as well as the ratio of AdoHcy/S-adenosylmethionine). Cells treated with neplanocin A also contained significant amounts of S-neplanocylmethionine, whereas cells treated with 1 or 2 showed no evidence of the formation of a similar metabolite. When neplanocin A and adenosine were incubated in cell lysates, rapid conversion to neplanocin D and inosine, respectively, were observed, illustrating the affinity of these nucleosides for cellular adenosine deaminase. In contrast, when 1 and 2 were incubated in cell lysates, no evidence for deamination was observed. These data illustrate that compounds 1 and 2 retain the inhibitory activity of neplanocin A toward cellular AdoHcy hydrolase, producing elevated cellular levels of AdoHcy. However, by removing the 4'-hydroxymethyl group from neplanocin A, analogs 1 and 2 are no longer substrates for adenosine deaminase and adenosine kinase.
Mol Pharmacol 1988 Jun
PMID:Effects of 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-adenine and -3-deazaadenine on the metabolism of S-adenosylhomocysteine in mouse L929 cells. 245 88

Human DNA was used to transform adenosine kinase (AK)-deficient BHK cells followed by selection of AK+ cells in medium containing alanosine, adenosine, and uridine (AAU medium). Twenty AAUr isolates were analyzed, and none of them contained AK activity. Several purine salvage enzymes were, however, found to be affected in these cells. The levels of hypoxanthine-guanine phosphoribosyltransferase and adenylosuccinate synthetase activities were elevated, while the adenylosuccinase activity was reduced. AAU-resistance may be explained by elevated activity of adenylosuccinate synthetase to overcome the alanosine block; thus AAUr cells were able to convert exogenous adenosine----inosine----hypoxanthine----IMP----AMPS----AMP. Moreover, these AAUr cells required exogenous purines for growth. HPLC analyses of endogenous nucleotide pools of AAUr cells showed that the levels of adenine nucleotides have diminished to less than 10% of the parental levels. These results suggest that the AAU-resistant mutation, which elicits pleiotropic phenotypes in BHK cells, affects an important component in the regulation of adenine nucleotide synthesis. By including erthyro-9-(2-hydroxy-3-nonyl)adenine in the AAU medium (renamed as AAUE medium) to block deamination of adenosine, AK+ BHK cells were isolated.
Somat Cell Mol Genet 1989 Mar
PMID:Imbalance of purine nucleotides in alanosine-resistant baby hamster kidney cells. 253 26

2',3'-Dideoxyinosine (ddlno) is a potent and selective inhibitor of human immunodeficiency virus in human lymphoid cells and monocytes/macrophages. Earlier studies [J. Biol. Chem. 263:15354 (1988)] showed that anabolism of ddlno in human lymphoid cells is mediated via an initial step of phosphorylation and subsequent amination to dideoxy-AMP via adenylosuccinate synthetase/lyase. Evidence was obtained that neither adenosine kinase nor deoxycytidine kinase is involved in the phosphorylation of this compound in human lymphoid cells. We now find that, in the presence of MgCl2, KCl, and inosine-5'-monophosphate as phosphate donor, purified cytosolic 5'-nucleotidase catalyzed the phosphorylation of ddlno. Although not phosphate donors, ATP, diadenosine tetraphosphate, and glycerate-2,3-bisphosphate stimulate this phosphorylation by the nucleotidase 4-5-fold. In addition to ddlno, the antiviral nucleoside analogs 2',3'-dideoxyguanosine and carbovir were substrates for this enzyme. The relative phosphorylation of these compounds varied with the concentration of the phosphate donor IMP. Approximate Km values of the nucleotidase for inosine, ddlno, dideoxyguanosine, and carbovir were, respectively, 3.4, 0.5, 0.9, and 1.7 mM. Although the substrate activity of dideoxynucleosides is inefficient, it appears likely that this nucleotidase is responsible for the metabolism of these compounds to their active nucleotides, yielding antiviral activity in human lymphoid cells.
Mol Pharmacol 1989 Aug
PMID:Phosphorylation of 2',3'-dideoxyinosine by cytosolic 5'-nucleotidase of human lymphoid cells. 254 85

The S-adenosylhomocysteine (SAH) hydrolase inhibitor adenosine dialdehyde was used in isolated guinea pig hearts to determine the contribution of the transmethylation pathway to cardiac adenosine formation. This inhibitor did not alter cardiac hemodynamics but effectively inhibited SAH-hydrolase activity under in vitro and in vivo conditions. In normoxic perfused hearts adenosine dialdehyde (10 microM) caused tissue levels of SAH to linearly increase at a rate of 160 pmol/g/min over 60 min. At the same time adenosine dialdehyde decreased release of adenosine into the coronary effluent perfusate by 16 pmol/min (34%). Hypoxic perfusion (30% O2) of guinea-pig hearts increased release of adenosine from 43 to 3700 pmol/min. However, rate of SAH formation in the presence of adenosine dialdehyde was only slightly enhanced from 160 to 200 pmol/g/min and adenosine dialdehyde did not significantly alter the hypoxia induced adenosine release. Since all experiments were performed in the presence of the adenosine deaminase inhibitor EHNA (5 microM) the results demonstrate: (1) the transmethylation pathway of the heart contributes one third to global cardiac adenosine production under normoxic conditions and provides a constant source of adenosine independent of tissue oxygenation. (2) The majority of SAH-derived adenosine is salvaged most likely via adenosine kinase. (3) The hypoxia induced adenosine production is predominantly derived from enhanced 5' AMP hydrolysis.
J Mol Cell Cardiol 1989 Aug
PMID:Contribution of S-adenosylhomocysteine to cardiac adenosine formation. 277 14

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.
Mol Biochem Parasitol 1988 Apr
PMID:Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani. 283 51


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