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Query: UNIPROT:P06889 (Mol)
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Adenosine aminohydrolase from calf intestinal mucosa is sensitive to changes in its environment produced by small mole fractions of dimethylsulfoxide (DMSO). At a mole fraction of 0.1 where the dielectric constant is lowered from that of 78 of neat water to about 76.5, Vmax was reduced by 65% and affinity for substrate (adenosine) and the two competitive inhibitors, insine and N6-benzyladenosine, was decreased markedly. However, this decreased affinity was such that Ki/Km remained virtually constant for both inhibitors. DMSO itself showed the kinetics of a mixed inhibitor with Ki decreasing with increasing mole fraction. This cosolvent also decreased the heat stability of the enzyme which suggests that enzyme conformation is altered by DMSO. Comparison of data in the presence of DMSO with previously obtained data with dioxane shows that heat stability as well as Vmax, at a given value of dielectric constant, is independent of the amount or nature of cosolvent used to achieve that dielectric constant. However, cosolvent induced changes in Ki indicate that colligative as well as dielectric constant effects contribute to the observed changes in kinetic behavior. These experiments may be considered as models for the behavior of enzymes in the medium of lowered dielectric constant expected in the vicinity of cytoplasmic membranes. The results indicate that in such an environment, adenosine aminohydrolase would be expected to be less efficient a catalyst, but equally susceptible to product inhibition, as compared to media of dielectric constant approaching that of water.
Mol Cell Biochem 1976 Aug 30
PMID:Cosolvent-buffer mixtures as models for the cytoplasmic mileu: the enzymology of adenosine aminohydrolase. 98 42

Caffeine potently inhibited forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex, with an IC50 of 21 +/- 3 microM. Because caffeine competitively blocks adenosine receptors, we examined whether the action of forskolin involved endogenous adenosine or whether caffeine was acting through some novel mechanism. Inhibition by caffeine was observed at all forskolin concentrations examined, although the degree of inhibition decreased at higher concentrations of forskolin. The effect of caffeine was not blocked by the presence of a phosphodiesterase inhibitor but was mimicked by several other methylxanthines. The most potent of these was 8-(p-sulfophenyl)-theophylline, which does not readily cross cell membranes, arguing for an extracellular site of action. Addition of either adenosine or the adenosine uptake blocker dipyridamole potentiated the forskolin response, suggesting that forskolin and adenosine act synergistically in increasing cyclic AMP accumulation. The nonxanthine adenosine receptor antagonist CGS 15943 potently blocked cyclic AMP responses to forskolin, adenosine, and combinations. 3-Isobutyl-1-methylxanthine potently blocked the response to adenosine but caused little or no inhibition of the response to forskolin. Adenosine deaminase (ADA) was added to eliminate contributions of endogenous adenosine. ADA inhibited the response to both adenosine and forskolin; however, 200 times as much enzyme was necessary to inhibit the forskolin response. Inhibition of added ADA with 2'deoxycoformycin dramatically increased the concentration of ADA required to inhibit the adenosine response, without altering the concentration required to inhibit the forskolin response. These results suggest that forskolin-stimulated cyclic AMP accumulation may be partially dependent on endogenous adenosine but that the inhibition observed with caffeine is not solely due to blockade of adenosine receptors.
Mol Pharmacol 1990 Nov
PMID:Is adenosine involved in inhibition of forskolin-stimulated cyclic AMP accumulation by caffeine in rat brain? 217 72

Adenosine deaminase is found primarily in the cytoplasm of many cell types. In the human erythrocyte, about 30 per cent of the total adenosine deaminase activity is membrane associated, and about two-thirds of this is inactivated by treatment of intact erythrocytes with the nonpenetrating reagent diazotized sulfanilic acid, without affecting lactate dehydrogenase, a soluble cytoplasmic enzyme. This indicates that within the cell membranes, the catalytic site of about two-thirds of the adenosine deaminase faces the external medium, i.e., ecto adenosine deaminase. Localization of adenosine deaminase activity at the cell membrane is demonstrated directly by electron microscopy by use of the substrate 6-Chloropurine ribonucleoside, which is dechlorinated by adenosine deaminase to produce Cl-, which is precipitated at its locus of formation by added Ag+, and the precipitated AgCl converted into the electron dense Ag0 upon exposure to light. From the Hydropathic Profile of the amino acid sequence of adenosine deaminase it is evident that there are two hydrophobic domains of sufficient length to span a biological membrane, and it is proposed that these domains could function to anchor the enzyme to the membrane. The importance of adenosine deaminase is indicated by the fatal immuno-deficiency which results from untreated genetic adenosine deaminase deficiency. It may be important to determine whether the amount of ecto adenosine deaminase activity is better suited to assess the clinical status of adenosine deaminase deficient patients that the currently used total cellular enzyme activity.
Mol Cell Biochem 1989 Apr 11
PMID:Ecto-enzyme activity of human erythrocyte adenosine deaminase. 277 Jul 11

Adenosine deaminase-deficient mutants of a mouse lymphoma cell line S49 have been isolated by a two-step selection process. In the first step, we derived mutant lines containing haploid levels of adenosine deaminase activity from wild-type cells. The selective medium contained tritiated deoxyadenosine, deoxycytidine, and deoxycoformycin. Wild-type cells were killed, presumably because of suicidal incorporation of tritiated deoxyadenosine via the adenosine deaminase pathway. The second step was to derive, from the partially deficient mutants, sublines that were virtually lacking adenosine deaminase, using tritiated deoxyadenosine and deoxycytidine. Four mutant clones were found to contain less than 5% of the enzyme activity of wild-type cells and virtually no immunoreactive adenosine deaminase protein. Northern blot analysis showed that the levels of adenosine deaminase mRNA were drastically reduced. Back-selection for adenosine deaminase-positive revertants can be accomplished by using a medium containing deoxyadenosine (as a sole source of purine), aminopterin, and thymidine or, alternatively, by using deoxyadenosine alone in a serum-free medium.
Somat Cell Mol Genet 1989 Sep
PMID:Isolation and characterization of S49 mouse lymphoma cell mutants deficient in adenosine deaminase. 278 37

The histamine-stimulated accumulation of [3H]cAMP (formed by prelabeling with [3H]adenine) was characterized pharmacologically in a vesicular preparation of guinea pig cortex. The H2 antagonist cimetidine maximally blocked 80% of the response, whereas only 45% of the response could be inhibited by H1 antagonists. A combination of H1 and H2 antagonists completely abolished the response. These and other findings show that both H1 and H2 receptors mediate the response, but 25% of the response may require simultaneous activation of both receptors. A role for adenosine as a mediator of the histamine response was investigated. Adenosine deaminase (EC 3.5.4.4., 2.5 units/ml) decreased basal [3H]cAMP levels, abolished the cimetidine-resistant component of the histamine response, and reduced maximal H1 antagonism of the histamine response to 30%. Treatment with a combination of adenosine deaminase and the calcium chelator EGTA (2 mM) appeared to eliminate the H1 component completely. Under these latter conditions only H2 receptors appeared to mediate the histamine response. Thus, both H1 and H2 receptors stimulate [3H]cAMP accumulation in the vesicular preparation, but the H1 response seems to require either concomitant adenosine or H2 receptor stimulation and may be calcium dependent. These findings differ from those found in broken cell membrane preparations, where only H2 receptors appear to be coupled to adenylate cyclase activation.
Mol Pharmacol 1988 Jan
PMID:Histamine receptors coupled to [3H]cAMP accumulation in brain: pharmacological characterization in a vesicular preparation of guinea pig cortex. 282 98

The involvement of adenosine in the coupling of insulin binding to action was investigated in rat adipocytes. Reduction of endogenous adenosine levels by treatment with adenosine deaminase (ADA) had no significant effect on either basal or maximally stimulated glucose transport, but reduced the insulin sensitivity of transport stimulation. Adenosine deaminase treatment also shifted the EC50 of H2O2 stimulation of transport from 0.13 mM to 0.30 mM, and the EC50 for insulin stimulation of protein synthesis from 0.40 +/- 0.06 ng/ml to 1.30 +/- 0.25 ng/ml. Adenosine appears to be acting through the pharmacological Ri adenosine receptor subtype. The mode of action of adenosine does not seem to involve inhibition of adenylate cyclase. Adenosine also influences the kinetics of insulin action. ADA treatment slows the onset of transport stimulation by a maximal insulin concentration (10 ng/ml). Increasing the hormone level to 100 ng/ml overcomes this slowing without increasing transport further. The deactivation of glucose transport following removal of insulin is accelerated by ADA treatment. Thus, adenosine is involved both in maintaining a high efficiency of an early step in the insulin signaling process and in maintaining optimal activity of the insulin-stimulated glucose transport system.
Mol Cell Endocrinol 1988 Nov
PMID:The role of adenosine in insulin action coupling in rat adipocytes. 285 Sep 47

Adenosine deaminase (adenosine aminohydrolase, EC3.5.4.4) has been purified from human erythrocytes using a simple chromatographic procedure. Purified enzyme was obtained from individuals who were homozygous for the principal isozyme (ADA 1) as well as from individuals who were heterogyzous for the major variant (ADA 2-1). Although ADA 1 and ADA 2-1 are electrophoretically distinguishable, they have many common physical and catalytic properties. No significant differences between the two isozymic forms were found in measurements of molecular weight, catalytic activity in the presence of various substrates and inhibitors, pH optimum, turnover number, and stability in conditions of both high and low pH. ADA 2-1 was, however, substantially less stable than ADA 1 with respect to thermal denaturation. These studies support the idea that adenosine deaminase activity in erythrocytes is lower in those individuals who possess the variant form of the enzyme.
Mol Cell Biochem 1982 Oct 18
PMID:Physical and catalytic properties of the isozymes of adenosine deaminase from human red blood cells. 714 44

Adenosine causes airway obstruction in asthmatics and smokers. Theophylline and cromolyn, drugs used to treat these patients, bind to human lung adenosine receptors (ARs). This study investigated whether A1ARs and/or A2ARs are functionally present in human lung and airways, and whether theophylline and/or cromolyn antagonize their function. Peripheral lung or airway fragments from 21 people were incubated for 15 min with (1) an A1AR agonist, N6-cyclopentyladenosine (CPA, 5 to 1,000 nM), or (2) an A2AR agonist, either 5'-N-ethylcarboxamido adenosine (NECA, 0.5 to 20 microM) or 2-[p-(2-carboxyethyl)-phenethyl amino]-5'-N-ethylcarboxamido adenosine (CGS 21680, 0.5 to 28 microM), in the presence of the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (50 nM) and/or (3) theophylline (1 mM) and/or (4) cromolyn (500 microM). Adenosine deaminase (2 U/ml) and the phosphodiesterase inhibitor Ro 20-1724 (2 mM) were present in all incubations. Cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. In peripheral lung, CPA did not change baseline or isoproterenol-stimulated cAMP content. However, both NECA (20 microM) and CGS 21680 (28 microM) significantly (P < 0.05) increased cAMP content 220 +/- 4% and 201 +/- 32%, respectively (mean +/- SEM). In airways, 20 microM NECA increased cAMP content 129 +/- 34%, and 28 microM CGS 21680 increased it 52 +/- 20% (both P < 0.05). In both peripheral lung and airway tissue, NECA-induced increase in cAMP was antagonized by theophylline (P < 0.05) but not cromolyn. The lungs of younger, nonsmokers had lower baseline cAMP content but did not respond differentially to A2AR stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1993 Aug
PMID:Effect of adenosine receptor ligands on cAMP content in human airways and peripheral lung. 839 27

In a retrospective study Adenosine deaminase (ADA), was assayed in 86 serum samples and 12 pleural fluid samples of patients with pulmonary tuberculosis. Serum and pleural fluid ADA levels were also examined in a group of 38 non-tuberculous patients with pleural effusion. Highly significant increases in both serum and pleural fluid ADA levels were noted in tuberculous patients when compared to both control enzyme cut-off values and non-tuberculous pleural effusion patients. Negative Ziehl-Nielsen staining for acid fast bacilli and Purified Protein Derivative skin test results were obtained in 32.3% and 11.1% of the examined patients respectively. All these patients showed significantly elevated serum or pleural fluid ADA levels. It is hereby suggested that serum and pleural fluid ADA levels can in conjunction with other tests, serve as a marker in patients of pulmonary tuberculosis.
Biochem Mol Biol Int 1997 Nov
PMID:Adenosine deaminase activity in serum and pleural effusions of tuberculous and non-tuberculous patients. 938 37

Adenosine deaminase (ADA) was isolated from small intestine of mice and purified to utmost homogeneity. SDS-PAGE of purified ADA gave a molecular weight of 41 kDa. Western blot analyses gave a single reactive band at 41 kDa and the other band was an associated ADA binding protein. The purified enzyme was more stable in the alkaline pH. The optimum pH and the pI values were about 7.0 and 4.96, respectively. Km values of the small intestinal ADA for adenosine and 2'-deoxyadenosine were 23 and 16 microM, respectively. Purine riboside was a competitive inhibitor with Ki of 5 microM, whereas 2'-3'-o-isopropylidene adenosine acted as an uncompetitive inhibitor (Ki 66 microM). Activity of ADA was inhibited by the presence of theophylline (-40%), caffeine (-30%), and L-cysteine (-50%). Significantly, Hg2+ (100 microM) inhibited 98% of the initial ADA activity. In addition, various purine analogs such as inosine, purine, alpha-adenosine and adenine showed variable inhibitions on the activity of ADA. Relative ADA activity towards 3'-deoxyadenosine and 6-chloropurine riboside was lower by 30% and 40%, respectively. However, the activity towards 2'-o-methyl adenosine was higher (30%) compared to the activity obtained using adenosine.
Mol Cell Biochem 2000 Jan
PMID:Purification and characterization of intestinal adenosine deaminase from mice. 1071 33


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