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

This study was carried out to evaluate the possible role of adenosine uptake and metabolism in mediating the inhibitory actions of this nucleoside on spontaneous mouse oocyte maturation. Uridine blocked 3H-adenosine uptake by oocyte-cumulus cell complexes (OCCs) and cumulus cell-enclosed oocytes (CEOs) by 82-85%, whereas uptake by denuded oocytes (DOs) was suppressed by 97%. Uridine had no effect on germinal vesicle breakdown (GVB) in CEOs when meiotic arrest was maintained with hypoxanthine or hypoxanthine plus adenosine but reversed the combined inhibitory action of these purines in DOs. Five of six adenosine analogs that bind to purinoceptors demonstrated meiosis-arresting activity but not in relation to their relative affinities for inhibitory or stimulatory adenosine receptors and only at high concentrations. Moreover, in DOs, uridine reversed the inhibitory effect of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, two receptor agonists that are poor substrates for adenosine-metabolizing enzymes. Results of experiments with adenosine kinase inhibitors showed that methylmercaptopurine riboside (MMPR) and tubercidin, but not 5'-amino-5'-deoxyadenosine, reversed meiotic arrest maintained by hypoxanthine +/- adenosine, but this required an additional inhibitory action on de novo purine synthesis. Inhibition of de novo purine synthesis alone was not sufficient because azaserine failed to reverse meiotic arrest. MMPR was a very potent meiosis-inducing agent, completely reversing meiotic arrest in CEOs and DOs in the presence of a variety of meiotic inhibitors. The adenosine deaminase inhibitor deoxycoformycin had opposite effects on oocyte maturation depending on the presence or absence of adenosine: the inhibitory action of hypoxanthine alone was bolstered, but the meiosis-arresting action of adenosine was reversed. These data therefore indicate that at low adenosine concentrations phosphorylation predominates, but at higher adenosine concentrations deaminated products contribute to the meiotic inhibition. This idea was borne out by the ability of inosine to mimic the synergistic interaction of adenosine with hypoxanthine. The action of adenosine is not due to deamination to inosine and conversion to nucleotides through the hypoxanthine salvage pathway because adenosine-mediated inhibition was not compromised in oocytes from mutant mice unable to salvage hypoxanthine.
Mol Reprod Dev 1999 Jun
PMID:Uptake and metabolism of adenosine mediate a meiosis-arresting action on mouse oocytes. 1033 59

The enzyme ribokinase phosphorylates ribose at O5* as the first step in its metabolism. The original X-ray structure of Escherichia coli ribokinase represented the ternary complex including ribose and ADP. Structures are presented here for the apo enzyme, as well as the ribose-bound state and four new ternary complex forms. Combined, the structures suggest that large and small conformational changes play critical roles in the function of this kinase. An initially open apo form can allow entry of the ribose substrate. After ribose binding, the active site lid is observed in a closed conformation, with the sugar trapped underneath. This closure and associated changes in the protein appear to assist ribokinase in recognition of the co-substrate ATP as the next step. Binding of the nucleotide brings about further, less dramatic adjustments in the enzyme structure. Additional small movements are almost certainly required during the phosphoryltransfer reaction. Evidence is presented that some types of movements of the lid are allowed in the ternary complex, which may be critical to the creation and breakdown of the transition state. Similar events are likely to take place during catalysis by other related carbohydrate kinases, including adenosine kinase.
J Mol Biol 1999 Jul 30
PMID:Induced fit on sugar binding activates ribokinase. 1043 99

A genetic approach has been exploited to investigate adenylate salvage pathways in the protozoan parasite Toxoplasma gondii, a purine auxotroph. Using a new insertional mutagenesis vector designed to facilitate the rescue of tagged loci even when multiple plasmids integrate as a tandem array, 15 independent clonal lines resistant to the toxic nucleoside analog adenine arabinoside (AraA) were generated. Approximately two-thirds of these clones lack adenosine kinase (AK) activity. Parallel studies identified an expressed sequence tag (EST) exhibiting a small region of weak similarity to human AK, and this locus was tagged in several AK-deficient insertional mutants. Library screening yielded full-length cDNA and genomic clones. The T. gondii AK gene contains five exons spanning a approximately 3 kb locus, and the predicted coding sequence was employed to identify additional AK genes and cDNAs in the GenBank and dbEST databases. A genomic construct lacking essential coding sequence was used to create defined genetic knock-outs at the T. gondii AK locus, and AK activity was restored using a cDNA-derived minigene. Hybridization analysis of DNA from 13 AraA-resistant insertional mutants reveals three distinct classes: (i) AK-mutants tagged at the AK locus; (ii) AK- mutants not tagged at the AK locus, suggesting the possibility that another locus may be involved in regulating AK expression; and (iii) mutants with normal AK activity (potential transport mutants).
Mol Biochem Parasitol 1999 Sep 20
PMID:Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus. 1051 76

Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus represents a promising target for rational design of antiparasitic compounds. In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T. gondii was overexpressed in E. coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of TgAK revealed Km values of 1.9 microM for adenosine and 54.4 microM for ATP, with a k(cat) of 26.1 min(-1). Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs. Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T. gondii AK. Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo.
Mol Biochem Parasitol 1999 Sep 20
PMID:Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase. 1051 77

Carbohydrate kinases frequently require a monovalent cation for their activity. The physical basis of this phenomenon is, however, usually unclear. We report here that Escherichia coli ribokinase is activated by potassium with an apparent K(d) of 5 mM; the enzyme should therefore be fully activated under physiological conditions. Cesium can be used as an alternative ion, with an apparent K(d) of 17 mM. An X-ray structure of ribokinase in the presence of cesium was solved and refined at 2.34 A resolution. The cesium ion was bound between two loops immediately adjacent to the anion hole of the active site. The buried location of the site suggests that conformational changes will accompany ion binding, thus providing a direct mechanism for activation. Comparison with structures of a related enzyme, the adenosine kinase of Toxoplasma gondii, support this proposal. This is apparently the first instance in which conformational activation of a carbohydrate kinase by a monovalent cation has been assigned a clear structural basis. The mechanism is probably general to ribokinases, to some adenosine kinases, and to other members of the larger family. A careful re-evaluation of the biochemical and structural data is suggested for other enzyme systems.
J Mol Biol 2002 Jan 18
PMID:Activation of ribokinase by monovalent cations. 1178 21

The activity of adenosine kinase is significantly impaired in tissues of diabetic rat. Changes in the activity of adenosine kinase were accompanied by alterations in its mRNA and protein level. These changes depended on insulin level and were not related to glucose level. During the first 7 h after insulin treatment the level of adenosine kinase mRNA, protein and enzymatic activity in kidneys, liver and heart returned to normal values. The observed relation between insulin and adenosine kinase expression level may suggest that insulin increases the rate of adenosine kinase gene transcription. Decreased activity of adenosine kinase was associated with elevated level of adenosine in diabetic tissues. On the 10th day after the STZ treatment there was a 3.5 and 2-fold increase in adenosine content in heart and liver, respectively. On the other hand, in diabetic kidney adenosine level was elevated only by 20%. Administration of insulin to diabetic rats resulted in a drop of adenosine to the level seen in normal heart and liver whereas, in kidneys the adenosine content was 50% lower than that observed under normal conditions. The time-dependent coarse of changes in adenosine level was different from that observed for adenosine kinase activity, what may suggest that other factors, possibly nucleoside transporters are also important for controlling adenosine level in the cell.
Mol Cell Biochem 2002 Jul
PMID:Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats. 1219 Jan 16

Evidence that the time course of insulin-induced changes in adenosine level in diabetic rats is different from that observed for expression of adenosine kinase prompted us to study the insulin effect on expression of nucleoside transporters in tissues of diabetic rats. RNase protection assay demonstrated that mRNA levels of equilibrative (rENT) and Na+-dependent nucleoside transporters (rCNT) were altered in diabetic tissues. The rENT1 mRNA level with respect to values obtained in age- and sex-matched nondiabetic rats was decreased by 45, 32, and 10% in diabetic heart, liver, and kidney, respectively. The level of rENT2 mRNA was lowered by 40% in diabetic kidney and heart, and by 24% in diabetic liver. Changes in the expression pattern of rCNT1 and rCNT2 in diabetic tissues differed significantly from that observed for rENT. The levels of rCNT1 and rCNT2 mRNA did not change significantly in diabetic kidney. In diabetic heart, the mRNA levels of rCNT1 and rCNT2 increased 1.7- and 2-fold, respectively. Changes in expression of nucleoside transporters were accompanied by alterations in adenosine content. Administration of insulin to diabetic rats resulted in a drop in adenosine concentration in examined tissues and return of the rCNT1, rCNT2, and rENT2 but not rENT1 mRNA levels to values observed in nondiabetic rats. In summary, these data demonstrate that insulin affects expression of nucleoside transporters in a cell-specific manner. We conclude that change in the expression level of the nucleoside transporters occurring in tissues of diabetic rat is an important factor influencing adenosine levels in the cell.
Mol Pharmacol 2003 Jan
PMID:The effect of insulin on expression level of nucleoside transporters in diabetic rats. 1248 39

New directions in computational methods for the prediction of protein function are discussed. THEMATICS, a method for the location and characterization of the active sites of enzymes, is featured. THEMATICS, for Theoretical Microscopic Titration Curves, is based on well-established finite-difference Poisson-Boltzmann methods for computing the electric field function of a protein. THEMATICS requires only the structure of the subject protein and thus may be applied to proteins that bear no similarity in structure or sequence to any previously characterized protein. The unique features of catalytic sites in proteins are discussed. Discussion of the chemical basis for the predictive powers of THEMATICS is featured in this paper. Some results are given for three illustrative examples: HIV-1 protease, human apurinic/apyrimidinic endonuclease, and human adenosine kinase.
Mol Biol Rep 2002 Dec
PMID:Future directions in protein function prediction. 1254 18

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.
J Biochem Mol Toxicol 2003
PMID:2'-Deoxyadenosine causes apoptotic cell death in a human colon carcinoma cell line. 1470 88

Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC(50) values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC alpha or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome.
Mol Cell Proteomics 2004 May
PMID:Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors. 1476 51


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