Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.4.4 (adenosine deaminase)
 5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To evaluate for selective toxicity of S-adenosylhomocysteine toward cultured lymphoblasts, cytotoxicity was correlated with S-adenosyl-L-homocysteine accumulation in cultured human B-lymphoblasts (MGL-8) and T-lymphoblasts (MOLT-4) during adenosine deaminase inhibition with EHNA. The addition of adenosine increased intracellular S-adenosylhomocysteine levels and decreased the growth of B-lymphoblasts, with an estimated ID50 of 50 micro M. These changes were enhanced by the addition of homocysteine thiolactone. The addition of deoxyadenosine, even with homocysteine thiolactone, had no effect in B-lymphoblasts. The addition of deoxyadenosine potently decreased the growth of T-lymphoblasts, with an estimated ID50 of 16 micro M, and increased intracellular S-adenosylhomocysteine concentrations. The changes were enhanced with the addition of homocysteine thiolactone. T-lymphoblasts cultured with adenosine showed only modest increases in intracellular S-adenosylhomocysteine levels but did have a substantial decrease in growth. These changes were not substantially modified by the addition of homocysteine thiolactone. S-adenosyl-L-homocysteine hydrolase activity did not correlate with cytotoxicity or S-adenosyl-L-homocysteine accumulation in B- or T-lymphoblasts. These data suggest that selective S-adenosyl-L-homocysteine accumulation and toxicity in B-lymphoblasts provide a potential mechanism for the B-lymphocyte defect in adenosine deaminase deficiency. The accumulation of S-adenosylhomocysteine in T-lymphoblasts and the associated cytotoxicity provide evidence to implicate this mechanism as contributing to the T-cell disorders in inherited or acquired adenosine deaminase deficiency.
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PMID:S-Adenosylhomocysteine accumulation and selective cytotoxicity in cultured T- and B-lymphocytes. 698 Feb 50

High levels of adenosine deaminase (ADA) activity have been associated with normal T cell differentiation and T cell disease, such as acute lymphoblastic leukemia; however, possible mechanisms controlling the level of this enzyme have not been explored. In this study, the properties and rate of turnover of ADA are compared in cultured human T and B lymphoblast cell lines. (1) Relative to B lymphoblasts, the level of ADA activity in extracts of T lymphoblast cell lines (MOLT-4, RPMI-8402, CCRF-CEM and CCRF-HSB-2) is elevated 7- to 14-fold and differs by 2-fold among the T-cell lines. (2) In T and B lymphoblast extracts, the enzyme is apparently identical based on Km for adenosine and deoxyadenosine, Ki for inosine, Vmax for adenosine, S20w, isoelectric pH, and heat stability. Further, by radioimmunoassay the quantity of ADA immunoreactive protein is proportional to the level of enzyme activity in all cell lines studied. (3) Using a purification and selective immunoprecipitation technique, the enzyme turnover could be assessed in cell lines labeled with [35S]methionine. The apparent rate of ADA synthesis, relative to total protein, is 2-fold faster in both T cell lines (RPMI-8402 and CCRF-CEM) than in the B cell lines (MGL-8 and GM-130). The apparent half-life (t1/2) for the enzyme degradation is 19 and 39 hr, respectively, for CCRF-CEM and RPMI-8402, while the t1/2 for both B cell lines is 7-9 hr. From the net rate of synthesis and degradation, the T cell lines exhibit a 6- and 12-fold difference in ADA turnover relative to B cells, consistent with the observed differences in enzyme activity. (4) The level of ADA (activity and/or protein) in cultured T or B lymphoblasts is not influenced by either substrates or products of the ADA reaction or an ADA inhibitor or a selected group of immunosuppressive drugs added to these cells in culture. These studies indicate that while ADA is apparently identical in all T and B lymphoblasts, alterations in both the rate of ADA synthesis and degradation lead to its accumulation and high steady-state level in T cells.
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PMID:Control of adenosine deaminase levels in human lymphoblasts. 698 Dec 87

The relationship between adenosine deaminase (ADA) and a human membrane thymus leukemia (HTL) antigen-detected by an antithymocyte serum was explored. A freeze-thaw extract of the T-cell-derived cell line, MOLT 4, was applied to an immunoabsorbant column of a rabbit antiserum to calf ADA. The bound MOLT 4 ADA was eluted with 6 M urea. The recovered ADA had a specific activity of 490 mumol of adenosine deaminated per min per mg protein, and the yield was 32%. No HTL antigenic activity was detected in the purified ADA. In addition, no ADA activity was detected in the unbound fraction containing the HTL antigenic activity, supporting the conclusion that ADA and HTL antigen are independent molecules. Affinity-purified anti-calf ADA was not cytotoxic for several HTL antigen-positive cells, including thymocytes, MOLT 4, and thymus-derived acute leukemia lymphoblasts.
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PMID:Relationship between adenosine deaminase and a human thymus leukemia antigen isolated from MOLT 4 cells. 719

The anti-human immunodeficiency virus agents 2',3'-dideoxyadenosine (ddAdo) and 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo) are rapidly converted, both in vitro and in vivo, to the corresponding inosine analogs by the widely distributed enzyme adenosine deaminase (EC 3.5.4.4). We have determined the effects of the potent adenosine deaminase inhibitor 2'-deoxycoformycin (2'-dCF) on ddAdo and 2'-beta-F-ddAdo metabolism in MOLT-4 cells and on ddAdo antiviral activity in the ATH8 test system. At levels as low as 5 nM in the incubation medium, 2'-dCF effectively blocks the extracellular deamination of both agents, thus permitting their rapid cellular uptake as the unchanged parent compounds, rather than as the less lipid-soluble 2',3'-dideoxyinosine or 2'-beta-fluoro-2',3'-dideoxyinosine. The result is a significant increase in intracellular levels of the pharmacologically active forms 2',3'-dideoxyadenosine-5'-triphosphate and 2'-beta-fluoro-2',3'-dideoxyadenosine-5'-triphosphate. The effect becomes maximal over the range of 50-250 nM 2'-dCF and declines to control levels when extracellular 2'-dCF levels exceed 1 microM. This decrease in ddAdo and 2'-beta-F-ddAdo phosphorylation with higher levels of the inhibitor appears to result from intracellular penetration of 2'-dCF and consequent inhibition of intracellular deamination, a critical step in the activation of both agents through the 5'-nucleotidase pathway. In anti-human immunodeficiency virus assays, a 2.2-fold increase in ddAdo antiviral potency was seen at 2'-dCF levels of 20 and 50 nM.
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PMID:Enhancement by 2'-deoxycoformycin of the 5'-phosphorylation and anti-human immunodeficiency virus activity of 2',3'-dideoxyadenosine and 2'-beta-fluoro-2',3'-dideoxyadenosine. 796 62

We have identified a 236-bp first intron segment of the mouse adenosine deaminase gene (ADA) that shares 71.1% identity with the human ADA thymic enhancer. This segment has the same natural orientation as the human enhancer and a relative location within the first intron very analogous to that of the human enhancer. Four highly conserved regions were defined within this segment, including a 72-bp region having 83.6% identity with a segment containing the critical human enhancer core. Several consensus binding sequences were also conserved within these regions. Transient transfection assays in human and murine T-cell lines revealed that a 1.8-kb murine genomic fragment harboring the 236-bp segment functions as a weak activator of both the human and mouse ADA promoters. In contrast, a 2.3-kb human enhancer fragment exhibited high-level activation in conjunction with either the human or mouse ADA promoter in both the MOLT 4 (human) and S49 (murine) T-cell lines. Interestingly, the murine ADA promoter is significantly stronger than the human promoter in driving cat expression in transient transfection assays in all the T-cell lines tested.
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PMID:Identification of a murine homolog of the human adenosine deaminase thymic enhancer. 856 89

The nucleoside analogue cordycepin (3'-deoxyadenosine, 3'-dA) is substantially more cytotoxic to terminal deoxynucleotidyl transferase positive (TdT+) leukemic cells than to TdT leukemic cells in vitro in the presence of an adenosine deaminase inhibitor, deoxycoformycin (dCF), and has been considered as a therapeutic agent for TdT+ leukemia. The intracellular metabolism of 3'-dA was examined with HPLC, and the mechanism of its anti-TdT+ leukemic activity was analyzed. In the presence of dCF (2.5 microM), TdT+ leukemic cells (N = 5) were sensitive to the cytotoxic effect of 3'-dA, whereas TdT (N = 6) cells were not. A high level of 3'-dA-5'-triphosphate (3'-dATP) formation was detected in TdT+ NALM-6 cells (67 pmol/10(6) cells) and TdT- K562 cells (49 pmol/10(6) cells) when cultured with 1 microM [3'-3H]-labeled 3'-dA. A substantial level of 3'-dATP was detected in TdT HUT-102 cells (27 pmol/10(6) cells), whereas the level of 3'-dATP in TdT+ MOLT-4 cells was low (0.3 pmol/10(6) cells). The mean IC50 values of 3'-dA against phytohemagglutinin (PHA)-activated and resting peripheral blood mononuclear cells (PBM) (N = 5) were 8 and 32 microM, respectively. There was a modest level of 3'-dATP (7 pmol/10(6) cells) in PHA-PBM, whereas a lower level of 3'-dATP was detected in resting PBM (2.5 pmol/10(6) cells). These data suggest that the presence of 3'-dATP is not sufficient for the antileukemic effect of 3'-dA, but that TdT positivity is essential, and that PBM are significantly less sensitive to the cytotoxicity of 3'-dA in vitro. Further development of 3'-dA as a potential antileukemic agent to treat patients with TdT+ leukemia is warranted.
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PMID:Antileukemic activity and mechanism of action of cordycepin against terminal deoxynucleotidyl transferase-positive (TdT+) leukemic cells. 1060 56

A non-radioactive procedure to measure the deoxycytidine kinase (dCK) activity in crude cell free homogenates was developed. 2-Chlorodeoxyadenosine (CdA) was used as the substrate for dCK and was separated from its product 2-chlorodeoxyadenosine-5'-monophosphate (CdAMP) by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 30 min. The minimum amount of CdAMP that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3h. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 microg/ml in the assay. An amount of 8 x 10(3) cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. CdA was not only converted to CdAMP but also to 2-chloroadenine and, surprisingly, also to 2-chlorodeoxyinosine, in MOLT-3 cells. The deamination of CdA was completely inhibited by deoxycoformycin, which clearly demonstrates that CdA is a substrate for adenosine deaminase.
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PMID:Determination of the deoxycytidine kinase activity in cell homogenates with a non-radiochemical assay using reversed-phase high performance liquid chromatography; Identification of a novel metabolite of 2-chlorodeoxyadenosine. 1513 10


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