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)

We describe a cytotoxic T lymphocyte-mediated cytotoxicity assay in which the release of a cytoplasmic enzyme, adenosine deaminase (ADA), instead of the widely used radioactive chromium is a measure of target lysis. In this enzyme-release assay the target is a mastocytoma P815-derived cell line, noted P815 ADA++, isolated by applying a selection procedure devised to specifically amplify the ADA gene. Gene amplification in P815 ADA++ was indeed demonstrated. Routine measurement of ADA activity from numerous supernatants is performed using a specific and sensitive colorimetric assay. The use of 96-well microtiter plates as well as of an automatic Multiscan spectrophotometer makes this measurement rapid and convenient. We show that this ADA-release assay is significantly more sensitive than the classical chromium-release test because of its consistently lower (5 to 10-fold) spontaneous release in 4 h, short-term cytotoxicity experiments. We also found that it is especially suited for the rapid detection, by visual screening, of rare, active killer clones among large, heterogeneous cytotoxic T lymphocyte populations. The assay could easily be adapted to other tumor targets (EL4, YAC-1, K562) of common use in studies involving immune lysis; indeed, the procedure of amplifying the ADA gene used in the isolation of the P815 ADA++ hyperactive line may be generally applied to these targets.
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PMID:Use of a P815-derived line with an amplified adenosine deaminase gene: an improved target for cellular cytotoxicity. 393 81

The adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), at low concentrations (less than 10 microM), enhances the inhibitory activity of adenosine against lymphocyte-mediated cytolysis (LMC) without itself being inhibitory. At higher concentrations, EHNA alone is inhibitory to LMC with an IC50 of 160 microM. This inhibition is reversible upon washout, appears to affect an early stage of the lytic process, and does not appear to involve changes in basal levels of cyclic AMP (cAMP), ribonucleoside 5'-triphosphate pool sizes, S-adenosylhomocysteine levels, or protein carboxymethylation. EHNA does enhance the cAMP response of cytolytic lymphocytes (CL) to activators of adenylate cyclase such as prostaglandin E1. EHNA inhibits lymphocyte high-affinity cAMP phosphodiesterase at immunosuppressive levels, exhibiting hyperbolic mixed-type inhibition (Ki = 83 microM, alpha = 0.47, beta = 0.18). Whereas inhibition of intralymphocytic ADA is complete at low concentrations (less than 25 microM) of EHNA, inhibition of LMC and intralymphocytic cAMP phosphodiesterase increases linearly with EHNA concentration to at least 200 microM. The presence of 200 microM EHNA during the centrifugation of mixtures of CL and EL4 leukemia target cells leads to increased CL cAMP levels. 2'-Deoxycoformycin, a more potent ADA inhibitor than EHNA, is not inhibitory to LMC and shows none of these cAMP-related effects. These results suggest that CL-target cell contact stimulates adenylate cyclase in the CL and that EHNA inhibits LMC due to its enhancement of this target cell-stimulated elevation of cAMP.
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PMID:Inhibition of lymphocyte-mediated cytolysis and cyclic AMP phosphodiesterase by erythro-9-(2-hydroxy-3-nonyl)adenine. 629 34