Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The aminopyrimidopyrimidine nucleoside 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (APP), which was previously shown to possess experimental antitumor and antiviral activity, was metabolized within WI-L2 human lymphoblastoid cells to a derivative identified as the beta-D-ribonucleotide (APP-MP). In a subline of WI-L2 cells deficient in adenosine kinase, this metabolite was not formed and APP was not cytotoxic, suggesting that APP is converted by adenosine kinase to its 5'-monophosphate. Because no evidence of di- or triphosphates was seen, the monophosphate appeared to be the active species. Treatment of WI-L2 or L1210 cells with APP (10 microM) for 30 min caused extensive depletion of both purine and pyrimidine ribonucleotides. Purine and pyrimidine deoxyribonucleotides were also depleted. Cells were not protected from the cytotoxicity of APP by hypoxanthine plus uridine, but uridine plus adenosine plus 2-deoxycoformycin gave considerable protection. This result was consistent with APP-MP acting as an inhibitor of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase, a hypothesis that was confirmed by preparing PRPP synthetase from Novikoff hepatoma cells; APP-MP was a noncompetitive inhibitor, with a Ki of 0.43 mM. APP-MP was found to accumulate in APP-treated cells to a concentration of almost 3 mM. The relevance of PRPP synthetase inhibition to the cytotoxic mechanism of APP is indicated by the fact that depletion of the PRPP pool was seen as early as 15 min after treatment, before any change was apparent in cellular levels of ATP or UTP. DNA synthesis was markedly suppressed within 30 min of APP treatment of WI-L2 cells, and a lesser degree of inhibition of RNA synthesis was apparent after 45 min.
Mol Pharmacol 1993 Aug
PMID:Inhibition of 5-phosphoribosyl-1-pyrophosphate synthetase by the monophosphate metabolite of 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine: a novel mechanism for antitumor activity. 768 45

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral agent with activity against herpes viruses and retroviruses, including human immunodeficiency virus, but its metabolism and mechanism of action remain unclear. We have isolated a human T lymphoid cell line (CEMr-1) that is resistant to the antiproliferative effects of PMEA. The antiviral effects of PMEA against human immunodeficiency virus-1 infection were also greatly reduced in CEMr-1 cells, compared with the parental cells. This mutant showed cross-resistance to the related acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)diaminopurine and 9-(2-phosphonylmethoxyethyl)guanine and the lipophilic prodrug bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine-( bispome-PMEA), as well as partial resistance to the purine nucleosides 2-chlorodeoxyadenosine, 2-fluro-9-beta-D-arabinosylfuranosyladenine, and adenosine, but did not show resistance to 2'-deoxyadenosine or 9-beta-D-arabinosylfuranosyladenine. We compared the uptake and metabolism of [3H]PMEA and [3H]-bispom-PMEA in the mutant and parental cells. The analysis of radioactive products by high pressure liquid chromatography revealed marked alterations in the ability of the mutant cell line to accumulate PMEA and its anabolites, compared with the parental cells. Accumulation of PMEA, PMEA monophosphate, and PMEA bisphosphate (major metabolites formed with either PMEA or bispom-PMEA) decreased by 50, 95, and 97%, respectively. Compared with the parental cells, the variant cells showed a approximately 7-fold increase in the rate of efflux of PMEA and a 2-fold decrease in the activity of adenylate kinase. In contrast, other enzymes of nucleotide metabolism, such as adenosine kinase, deoxycytidine kinase, and 5-phosphoribosyl-1-pyrophosphate synthetase, showed no significant change in the two cell lines. Overall, these results suggest that the mutation in this resistant cell line is of a novel type, involving an alteration in the cellular efflux of PMEA as the major basis for the resistant phenotype.
Mol Pharmacol 1995 Feb
PMID:A human T lymphoid cell variant resistant to the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine shows a unique combination of a phosphorylation defect and increased efflux of the agent. 787 49

Evaluation of enzyme activities involved in nucleotide metabolism and adenosine production within different cell types can provide important information on their contribution to the overall metabolism of the heart. The following enzyme activities were determined: adenosine kinase (AK), adenosine deaminase (ADA), S-adenosylhomocysteine hydrolase (SAHH), purine nucleoside phosphorylase (PNP), AMP deaminase (AMPD), membrane 5'nucleotidase (M5'N), AMP specific (AC5'N) and IMP specific (IC5'N) cytosolic 5'nucleotidases in (1) rat heart (n = 5), (2) rat cardiomyocytes obtained by collagenase digestion (n = 5), (3) human heart (n = 6) obtained from explants or papillary muscles collected during heart transplantation or mitral valve replacement, and (4) human umbilical cord endothelial cells in primary culture (n = 4). In the human heart, activities (mumol/min/g wet weight) were as follows: AK (0.14 +/- 0.01), ADA (0.46 +/- 0.03), SAHH (0.001 +/- 0.0003), PNP (0.43 +/- 0.08), AMPD (0.41 +/- 0.05), M5'N (1.75 +/- 0.12), IC5'N (0.21 +/- 0.03) and AC5'N (0.11 +/- 0.02). These enzyme activities were lower than those determined in the rat heart with the exception of AC5'N and IC5'N which were equal. The most prominent difference observed was for AMPD and M5'N which were nine and five-fold more active in the rat heart. Rat cardiomyocyte enzyme activities were comparable to those measured in whole rat heart with the exception of ADA (six-fold lower) and PNP (16-fold lower). Endothelial cell activities were notably different from those in the human heart particularly in the case of SAHH (nine-fold higher) and PNP (16-fold higher).(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1994 Nov
PMID:Nucleotide and adenosine metabolism in different cell types of human and rat heart. 789 72

(1'R,2'S,3')-9-(2',3'-Dihydroxycyclopentan-1'-yl)adenine (DHCaA), (1'R,2'S,3'R)-9-(2',3'-dihydroxycyclopentan-1'-yl)-3-deazaadenine (3-deaza-DHCaA), (4'R)-4'-methyl-DHCaA, and (4'R)-4'-vinyl-DHCaA, which are analogs of the carbocyclic nucleoside aristeromycin, were synthesized earlier by our laboratory and were shown to be potent inhibitors of purified bovine liver S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1). In the present study, these analogs were shown to produce rapid (within 15 min) and concentration-dependent (0.03-10 microM) inhibition of AdoHcy hydrolase in cultured murine L929 cells [relative order of inhibitory activity, DHCaA = 3-deaza-DHCaA >> (4'R)-4'-vinyl-DHCaA = (4'R)-4'-methyl-DHCaA]. The relative potencies of these inhibitors on the L929 AdoHcy hydrolase were consistent with their inhibitory effects on the recombinant forms of rat liver and human placental enzymes. This inhibition of L929 cellular AdoHcy hydrolase persisted for up to 48 hr. The inhibition of the L929 AdoHcy hydrolase resulted in a significant increase in the cellular concentrations of AdoHcy, whereas the cellular S-adenosylmethionine (AdoMet) levels remained relatively constant, thereby elevating the AdoHcy/AdoMet ratios. Maximum increases in AdoHcy levels and AdoHcy/AdoMet ratios occurred within 6 hr of exposure to the inhibitors and persisted for at least 24 hr. At a concentration of 1 microM, DHCaA and 3-deaza-DHCaA increased AdoHcy/AdoMet ratios to approximately 0.8 (after 24 hr of exposure to the inhibitors), whereas (4'R)-4'-vinyl-DHCaA and (4'R)-4'-methyl-DHCaA elevated AdoHcy/AdoMet ratios to approximately 0.15, compared with control levels of 0.05. Treatment of L929 cells with concentrations of DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA up to 10 microM did not result in changes in cellular levels of endogenous nucleotides (e.g., CTP, UTP, ATP, and GTP). In contrast, cells treated with 10 microM aristeromycin for 6 hr contained reduced cellular levels of CTP, ATP, and GTP and significant levels of aristeromycin triphosphate and a GTP metabolite of this carbocyclic nucleoside. These data clearly show that the 4'-modified analogs [DHCaA, 3-deaza-DHCaA, (4'R)-4'-vinyl-DHCaA, and (4'R)-4'-methyl-DHCaA] retain inhibitory activity toward cellular AdoHcy hydrolase, causing elevated levels of AdoHcy and elevated AdoHcy/AdoMet ratios. However, these analogs are devoid of substrate or inhibitory activity toward cellular adenosine kinase. In addition, aristeromycin is rapidly metabolized in murine L929 cell lysates, i.e., > 60% of the aristeromycin had been metabolized in 6 hr. In contrast, neither DHCaA nor 3-deaza-DHCaA showed any decrease in concentration after incubation with cell lysates for up to 6 hr.
Mol Pharmacol 1993 Jun
PMID:Effects of 4'-modified analogs of aristeromycin on the metabolism of S-adenosyl-L-homocysteine in murine L929 cells. 831 27

Monophosphoryl lipid A (MLA), a derivative of the minimal substructure of lipopolysaccharide (lipid A) possesses immunomodulatory activity of the parent lipid A yet enjoys reduced toxicity. It has previously been reported that pretreatment with MLA reduces myocardial infarct size and stunning in dogs following ischemia and reperfusion. The aim of this study was to evaluate the ability of monophosphoryl lipid A (MLA) to preserve global cardiac function and peripheral hemodynamics in a rabbit model of prolonged regional ischemia (90 min), and reperfusion (6 h). An evaluation of potential mechanisms by which MLA may preserve cardiac function was also undertaken. Single dose pretreatment with MLA (35 micrograms/kg i.v.) 24 h prior to ischemia resulted in significant improvement in left ventricular developed pressure, dP/dt, rate-pressure product and mean arterial pressure during reperfusion (P < 0.05 v control). Although in this model of prolonged ischemia MLA pretreatment did not reduce infarct size (54.5 +/- 11.4% in control v 63.3 +/- 8.3% in MLA, P = N.S.), evaluation of myocardial adenylate and adenosine catabolite pools at the end of ischemia indicated a preservation of ATP and ADP and a decreased production of downstream adenosine catabolites including inosine, xanthine and uric acid. Adenosine kinase, but not 5'-nucleotidase (5'-NTase) or adenosine deaminase activity determined following reperfusion was 76% and 60% higher (P < 0.05) in non-risk and post-ischemic myocardium of MLA pretreated rabbits compared with controls. Although there was a trend toward lower tissue myeloperoxidase activity in post-ischemic myocardium from treated rabbits, the results were not significantly different from control animals. These results suggest that a 24-h pretreatment with MLA, without further treatment during ischemia or reperfusion was associated with: (1) preservation of global myocardial function during reperfusion; (2) preservation of myocardial high energy adenylates and reduced formation of adenosine catabolites during ischemia; (3) elevated myocardial adenosine kinase activity. Increased recycling of adenosine to phosphorylated nucleotides may result from MLA's affect on adenosine kinase, which could explain the drugs effect on adenylate and adenosine metabolite pools.
J Mol Cell Cardiol 1996 Jan
PMID:Preservation of global cardiac function in the rabbit following protracted ischemia/reperfusion using monophosphoryl lipid A (MLA). 874 27

Formycin A augments insulin release evoked by glucose (5.6 mm or more), this effect not being rapidly reversible. The mechanism responsible for the insulinotropic action of formycin A was investigated in isolated pancreatic islets. It could not be ascribed to facilitation of glucose metabolism. On the contrary, formycin A inhibited glucose oxidation, lowered ATP content, and impaired glucose-stimulated protein biosynthesis. The insulinotropic action of formycin A was apparently attributable to its conversion to formycin A 5'-triphosphate, both this process and the secretory response to formycin A being abolished by the inhibitor of adenosine kinase 5-iodotubercidin. In agreement with the latter view, adenosine receptor antagonists such as 8-cyclopentyl-1, 3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine failed to suppress and, instead, augmented the insulinotropic action of formycin A. Unexpectedly, however, formycin A failed to decrease 86Rb efflux, this coinciding with a low efficiency of formycin A 5'-triphosphate to inhibit KATP-channel activity in excised membranes and with the fact that formycin A increased gliben-clamide-stimulated insulin release. The secretory response to formycin A represented a Ca2+-dependent process suppressed in the absence of extracellular Ca2+ or presence of verapamil and associated with an increased net uptake of 45Ca. Nevertheless, the view that formycin A exerts any major effect upon intracellular Ca2+ redistribution, protein kinase C activity, or cyclic AMP net production also met with objections such as the minor secretory effect of formycin A in islets exposed to a high concentration of K+ in the presence of a diazoxide analog, the resistance of formycin A insulinotropic action to bisindolylmaleimide, the poor increase of cyclic AMP content in formycin A-stimulated islets, and the pronounced enhancement by forskolin or theophylline of insulin release from islets exposed to formycin A. It is concluded, therefore, that the mechanism of action of formycin A in the pancreatic beta-cell remains to be elucidated.
Biochem Mol Med 1996 Feb
PMID:The riddle of formycin A insulinotropic action. 881 26

Purified adenosine kinase (AK) from Syrian hamster and bovine liver was examined for the presence of adenosine (Ad)-AMP exchange activity. The enzyme from both sources, in addition to catalyzing the conventional ATP-dependent phosphorylation of adenosine, supported an Ad-AMP exchange reaction that required ADP. Under optimal conditions both these reactions were found to occur at comparable rates. Several observations strongly indicate that the Ad-AMP exchange activity is an integral part of AK and it is likely associated with its catalytic mechanism. These observations include: (i) Both AK and Ad-AMP exchange activities show a nearly complete dependence upon the presence of pentavalent ions such as phosphate, arsenate or vanadate for catalysis; (ii) Both activities show similar heat-lability and inhibition by 5-iodotubercidin (5-ITu); (iii) In a Chinese hamster cell mutant resistant to adenosine analogs that lacked AK activity, the Ad-AMP activity was also found to be absent. The presence of a phosphoryl-enzyme intermediate, or any exchange between free 32Pi and any of the reactants, however, was not detected under the reaction conditions. Some implications of these observations regarding the catalytic mechanism of AK are discussed.
Biochem Mol Biol Int 1996 Jun
PMID:Adenosine-AMP exchange activity is an integral part of the mammalian adenosine kinase. 882

The enzyme adenosine kinase (AK) has been purified to homogeneity from Syrian hamster and bovine livers. The purified enzymes from both these sources have a Mr of approximately 38 kDa, as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis. A novel characteristic of AK observed here is that its catalytic activity shows a nearly complete dependence upon the presence of pentavalent ions such as phosphate (P(i)), arsenate or vanadate. Maximal AK activity was observed in the presence of either 2-3 mM P(i), or 5-10 mM arsenate, or 10-20 mM vanadate. A low basal level of AK activity (1-5% of maximal) observed in the absence of these ions is attributed to P(i) contamination in the adenine nucleotides preparations. The presence of P(i) had no effect on the K m for ATP (0.4 mM), but it markedly increased the affinity of the enzyme for adenosine. The K(m) of AK for adenosine in presence of 0, 0.1 mM and 2 mM P(i) was estimated to be 1.4 mu M, 0.77 mu M and 0.095 mu M, respectively. Free P(i) showed no exchange with any of the reactants during the assay conditions, and its presence had no effect on the thermostability of the enzyme. These observations suggest that the pentavalent ions such as phosphate may be playing an important role in the enzyme's catalytic mechanism by facilitating either binding of adenosine to the enzyme or in the formation of an enzyme-ATP-adenosine complex.
Biochem Mol Biol Int 1996 Apr
PMID:Pentavalent ions dependency of mammalian adenosine kinase. 913 58

The objective of the present study was to establish the optimal combination of inhibitors of adenosine metabolism and nucleotide precursors resulting in long-term increase in endogenous adenosine concentration without adverse metabolic consequences in non-ischemic cardiomyocytes and endothelial cells. Cardiomyocytes and endothelial cells were isolated after collagenase digestion of the rat heart. Freshly isolated cardiac myocytes or cultured endothelial cells were incubated for up to 8 h with no inhibitors or substrates or with various combinations of adenosine deaminase inhibitor: 5 micron M erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), adenosine kinase inhibitors: 10 micro M 5'-iodotubercidin (ITu) or 10 micro M 5'-aminoadenosine (AA) and nucleotide precursors: 100 micro M adenine, 2.5 mm ribose and 5 mm inorganic phosphate. Nucleotide, nucleoside and base concentrations were evaluated at the end of the incubation by HPLC in cardiomyocyte or endothelial cells extracts and in incubation media. Adenosine content in cardiomyocyte suspension was enhanced after 3 h incubation in the presence of ITu+EHNA as compared to EHNA alone (2.8+/-0.2 v 0.9+/-0.2 nmol/mg protein, respectively). ATP decreased from an initial value of 22.7+/-0.7 nmol/mg protein to 18.9+/-0.7 in the presence of ITu+EHNA, while ATP was maintained at 21.8+/-0.7 nmol/mg protein with EHNA. With adenine+ITu+EHNA, the changes were similar to those observed with ITu+EHNA. However, with ribose+adenine+ITu+EHNA, ATP increased to 25. 8+/-1.2 nmol/mg protein and adenosine concentration was elevated to 3.9+/-0.3 nmol/mg protein. Similar results were observed if AA was used instead of ITu to inhibit adenosine kinase. All the changes were maintained after 8 h of incubation. Adenosine content was increased in endothelial cells incubated with ITu+EHNA to 3.1+/-0.4 nmol/mg protein as compared to 1.1+/-0.2 nmol/mg protein with EHNA alone after 3 h, while ATP decreased (18.1+/-1.1 v 22.0+/-1.4 nmol/mg protein with EHNA+ITu or EHNA, respectively). In the presence of adenine+ITu+EHNA, adenosine content increased after 3 h to 6.5+/-0.9 nmol/mg protein while ATP was elevated to 26.1+/-0.8 nmol/mg protein. Additional presence of ribose was without effect. No changes in adenylate energy charge were observed in cardiomyocytes or endothelium under any conditions studied. Inhibition of adenosine kinase and adenosine deaminase caused a decrease in ATP together with increased adenosine content both in endothelial cells and cardiomyocytes. However, the addition of adenine (endothelial cells) or adenine with ribose (cardiomyocytes) together with inhibitors of adenosine metabolism protected cells from ATP depletion and further increased adenosine concentration.
J Mol Cell Cardiol 1998 Mar
PMID:Adenine/ribose supply increases adenosine production and protects ATP pool in adenosine kinase-inhibited cardiac cells. 951 42

Adenosine levels present in the interstitial fluid and coronary effluent of the aged heart exceed those of the young adult heart. The present study investigated mechanisms in the Fischer 344 rat heart which may be responsible for the observed differences. (1) Total production of adenosine was determined in isolated perfused hearts by measuring coronary effluent adenosine content while inhibiting adenosine deamination and rephosphorylation with erythrohydroxy-nonyladenosine (EHNA) and iodotubercidin (ITC), respectively. Total adenosine production was similar in both young (3-4 month) and aged (20-21 month) hearts at 31.8 +/- 6.6 and 38.4 +/- 3.3 nmol/min/g dry wt, respectively. However, stimulation with the beta-adrenergic agent, isoproterenol, elicited a significantly greater increase in adenosine production in the young vs. aged heart. (2) Adenosine transport was evaluated in isolated perfused hearts by determining 14C uptake by the myocardium after 20 min of 14C-adenosine perfusion. Adenosine uptake in the agent-free heart was found to be decreased 17 to 25% in aged compared to young adult hearts. (3) Adenosine transport characteristics were determined with nitrobenzylthioinosine saturation-binding studies in ventricular membrane preparations. The Bmax values were significantly lower in aged than young adult hearts (140.2 +/- 1.5 fmol/mg and 191.9 +/- 2.3 fmol/mg in aged and young hearts, respectively) indicating a decreased number of transporter sites in the aged heart. However, the values for Kd were decreased with aging, suggesting an increase in the affinity of the transporter for adenosine in the aged vs. young adult heart. (4) The activities and kinetics of adenosine kinase were determined in homogenates of aged and young adult ventricular myocardium. No statistical difference was found between the two activities. Taken together these results suggest that increased interstitial adenosine levels in the aged heart result from decreased uptake of adenosine by the ventricular myocardium.
J Mol Cell Cardiol 1999 Feb
PMID:Effect of aging on myocardial adenosine production, adenosine uptake and adenosine kinase activity in rats. 1009 52


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