Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.4.17 (adenosine deaminase)
 5,206 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Deoxycoformycin (dCF) is a specific inhibitor of adenosine deaminase (ADA). Rat hepatoma cells deficient in adenosine kinase and growing on adenosine as the sole carbon source are sensitive to the lethal action of dCF. Mutants resistant to dCF arise spontaneously with a frequency of 1.7 x 10(-6). This frequency is increased to 2.6 x 10(-5) by prior mutagenesis with ethyl methane sulfonate. Initially, dCF-resistant cell lines have 3-10 times the level of adenosine deaminase when compared to sensitive parental cells. Subsequent selection of mutants resistant to increased concentrations of dCF results in cells with a 15- to 30-fold increase in ADA levels. Quantitative immunoprecipitation tests indicate that the increase in enzyme activity in one line tested is due to an increase in the number of ADA molecules. These dCF' cell lines may serve as a model system to study the human disease state, hereditary hemolytic anemia, which is associated with increased levels of ADA.
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PMID:Isolation of deoxycoformycin-resistant cells with increased levels of adenosine deaminase. 698 55

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.
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PMID:Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase. 716 15

Reaction of formaldehyde with DNA in vitro produces a variety of adducts among which are observed the cross-link di-(N(6)-deoxyadenosyl)methane (dAdo-CH 2-dAdo, 1) and the hydroxymethyl adduct N(6)-hydroxymethyl-dAdo (N(6)-HOCH 2-dAdo, 2). While the structures of these adducts have been known for decades, there have been no reports of their formation in vivo. Formaldehyde is released during intracellular metabolism of carcinogenic N-nitrosomethyl compounds such as N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), but DNA adducts formed by this pathway have not been previously characterized. In this study, we addressed these questions by developing highly sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring methods for quantitation of adducts 1 and 2, the latter as N(6)-methyl-dAdo ( 3). Considerable effort was devoted to the problem of artifactual formation of 1, which can occur during storage of DNA samples by reaction of dAdo with 2. This problem was solved by the addition of adenosine deaminase during the DNA hydrolysis step, thus removing dAdo as a reactant. The instability of adduct 2 was another potential block to analysis, and this was solved by converting it to 3 with NaBH 3CN. Separate aliquots of DNA were analyzed for adducts 1 and 2, using the [(15)N]-labeled adducts as internal standards. The application of these methods to rat hepatic DNA to which adducts 1 and 3 were added demonstrated accuracy and precision. The detection limits for adducts 1 and 3 were 1-4 adducts per 10 (9) nucleotides using 100-150 microg of DNA. The method was applied to analyze hepatic and pulmonary DNA from rats treated with NDMA and NNK. The results clearly demonstrated the dose-dependent presence of N(6)-HOCH 2-dAdo ( 2) in all DNA samples. The cross-link adduct dAdo-CH 2-dAdo ( 1) was observed in hepatic DNA of NNK-treated rats, with lower amounts in pulmonary DNA. Levels of these adducts were generally less than those of DNA adducts formed by the classical diazohydroxide pathway of nitrosamine metabolism. The results of this study demonstrate for the first time the presence of formaldehyde DNA adducts in tissues of rats treated with carcinogenic nitrosamines and suggest that these adducts may play a role in cancer induction by these compounds.
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PMID:Development of liquid chromatography electrospray ionization tandem mass spectrometry methods for analysis of DNA adducts of formaldehyde and their application to rats treated with N-nitrosodimethylamine or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. 1767 14