Decrease of serum adenine nucleotide hydrolysis in an irritant contact dermatitis mice model: potential P2X7R involvement.

Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.

Time-dependent loss of adenosine 5'-monophosphate deaminase activity may explain elevated adenosine 5'-triphosphate levels in senescent erythrocytes.

Senescent erythrocytes from rabbits were previously shown to have elevated levels of adenine nucleotides. The present study documents that aged red blood cells have a normal synthetic capacity for adenine nucleotides, as indicated by normal levels of adenosine kinase. However, senescent erythrocytes do have decreased levels of adenosine 5'-monophosphate deaminase, the critical enzyme involved in degrading adenine nucleotides. These circumstances of a normal synthetic capacity in the presence of decreased catabolic ability were observed previously in a human genetic deficiency of adenosine 5'-monophosphate deaminase; the red blood cells in these patients accumulate adenosine 5'-triphosphate as do senescent erythrocytes in rabbits.

AMP deaminase as a cell-age marker in transient erythroblastopenia of childhood and its role in the adenylate economy of erythrocytes.

Erythrocytes from 11 patients with presumptive diagnoses of transient erythroblastopenia of childhood were evaluated retrospectively (six) or prospectively (five) for a possible relationship between erythrocyte adenosine 5'-monophosphate aminohydrolase, adenylic acid deaminase (AMP deaminase) activity and intracellular concentrations of adenine nucleotides. Older red blood cell (RBC) cohorts in these patients consistently exhibited significantly decreased activities of AMP deaminase (approximately 5% to 70% of normal control mean) in association with increased concentrations (up to threefold) of adenosine triphosphate (ATP) and total adenine nucleotides. We postulate that the latter is a direct consequence of the former, since diminishing AMP deaminase activity in aging cells should reduce the drain on the adenine nucleotide pool imposed by irreversible deamination of AMP to inosine 5'-monophosphate. Consistent reductions in AMP deaminase activity indicate that this enzyme should also serve as a reliable marker of mean RBC age useful in diagnostic confirmation of transient erythroblastopenia. The observed increases in ATP and total adenine nucleotides in older RBCs require a reevaluation of the traditional view that age-related losses of these compounds mediate the ultimate demise of senescent erythrocytes. Similar alterations in the balance of degradative and salvage pathways in RBC nucleotide metabolism may also underlie certain cases of so-called "high ATP syndrome."

Radioisotopic assay for erythrocyte adenosine 5'-monophosphate deaminase.

Adenosine-5'-monophosphate deaminase is a critical enzyme in the regulation of adenine nucleotide levels in the erythrocyte. The routine examination of this enzyme in crude hemolysates is difficult with the commonly used assay which monitors ammonia generated by the deamination reaction. This report details a radioisotopic assay for AMP deaminase which allows separation of the [14C]inosine 5'-monophosphate product from the [14C]adenosine 5'-monophosphate substrate by ion-exchange chromatography at pH 2.2. The radioisotopic assay is linear with respect to time and enzyme concentration over a considerable range and thereby significantly simplifies the monitoring of crude or dilute enzyme preparations.

Activation by calcium of AMP deaminase from the human red cell.

We have investigated the effects of Ca2+ on AMP deaminase from human red cells. At variance with the other known modulators, Ca2+ increased the apparent affinity for AMP without modifying the characteristic positive cooperativity of the enzyme towards the substrate. Ca2+ sensitivity was not modified by dialysis, but dilution of the haemolysate produced an activation of the enzyme similar to that induced by Ca2+. Simultaneously, the Ca2+ dependence was lost. The sensitivity to other modulators, such as ATP, diphosphoglycerate or phosphate, was not modified by dilution. Partial purification of the enzyme produced the same effects as haemolysate dilution. These results may be interpreted to mean that Ca2+ acts by antagonizing an endogenous inhibitor present in red cell lysates.

[AMP-deaminase activity of circulating leukocytes in the human]. / AMP-dezaminaznaia aktivnost' leikotsitov perifericheskoi krovi cheloveka.

It is shown that the AMP-deaminase activity in leucocytes of the human peripheric blood in contrast with the enzyme from erythrocytes manifests its activity only if it is isolated in the presence of K+ or Na+ ions. Pi and GTP being inhibitors of the enzyme in different tissues including erythrocytes do not alter the AMP-deaminase activity in leucocytes. 3,3',5-triiodothyracetic acid markedly decreasing the AMP-deaminase activity of leucocytes does not affect the enzyme activity in the hemolyzate of erythrocytes. The results obtained have shown that the AMP-deaminase activity in leucocytes of the human peripheric blood possesses some regulatory properties differing from those of the enzyme in erythrocytes.

Kinetics of the interactions of the chicken erythrocyte AMP deaminase with anthraquinone compounds.

Alizarine sulfonate, the anthraquinone containing both sulfonate and hydroxyl groups, showed an activating and inhibitory effect on the chicken erythrocyte AMP deaminase (EC 3.5.4.6). The cooperative effect of AMP, analyzed in terms of Hill coefficient, was decreased from 2.4 to 1.1 with the increase in the dye concentration, suggesting the dye as an allosteric activator of the enzyme. However, alizarine sulfonate acted as a mixed type inhibitor in the presence of higher level of AMP. The action of alizarine sulfonate can be accounted for by assuming that the dye binds to the enzyme at the allosteric-activating sites with a broad specificity toward nucleotide binding, and further at the specific inhibitory sites.

Deoxycytidylate deaminase in the diagnosis of acute myocardial infarction.

The diagnostic value of deoxycytidylate deaminase (dCMP) in the diagnosis of acute myocardial infarction (AMI) was examined in 31 healthy controls and 32 patients with proven AMI. Its specificity in normal controls was 93.5%. Its highest levels were measured on the 3rd to 5th post-infarct day, when its sensitivity reached 97.5%. Its diagnostic performance was similar to that of SGOT, total LDH1/LDH2 ratio greater than 1, total CPK and CPK-MB. Its peak levels showed a highly significant correlation (p less than 0.001) with those of the above enzymes. The measurement of dCMP may be a valuable complement in the diagnosis of AMI since this enzyme is not affected by lung infarction or hemolysis. Moreover, since it expresses tissue regeneration rather than destruction it may aid in the assessment of various interventions designed for the promotion of myocardial healing process.

Kinetics of the inhibition by naphtholsulfonate compounds of AMP deaminase from chicken erythrocytes.

The effect of a variety of naphthalene sulfonate compounds on the chicken erythrocyte AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) reaction was analyzed kinetically. Of the naphthalene sulfonate derivatives tested, the compounds with hydroxyl, sulfonate and nitrogen groups such as amino, anilino or azo groups showed an inhibitory effect. The cooperative effect of AMP, analyzed in terms of Hill coefficient, was increased from about 2 to 4 and the maximal velocity was unchanged with the addition of these compounds, suggesting the ligands as an allosteric inhibitor of the enzyme. The inhibition of AMP deaminase by naphtholsulfonate compounds can be qualitatively and quantitatively accounted for by the Monod-Wyman-Changeux model. Theoretical curves yield a satisfactory fit of all experimental saturation and inhibition curves, assuming four binding sites for AMP and the inhibitor, and various KT(I) values. The structure-activity analysis of the interaction of the naphtholsulfonate compounds with AMP deaminase has demonstrated that the affinity of the enzyme for naphtholsulfonates as the inhibitors is correlated with electronic properties of the nitrogen atoms attached to naphthalene moiety: the delocalization of lone electron pair on nitrogen through naphtholsulfonate group makes the compound less basic, resulting in more tight binding of the ligand to the enzyme. Introduction of hydrophobic group to naphtholsulfonate moiety increases the binding affinity for the enzyme, and of the inhibition. These results suggest the location of hydrophobic regions as the allosteric inhibitory sites of the enzyme for the binding of naphtholsulfonate compounds.

Distribution of AMP deaminase isozymes in various human blood cells.

Column chromatographic, electrophoretic and immunological studies showed the distribution of AMP deaminase isozymes in human blood cells as follows; isozyme E1 in erythrocyte, E2 in granulocyte, L in mononuclear cells, platelets and T-lymphoblast, and probably E1-L hybrid sets in B-lymphoblast.

AMP deaminase isozymes in human blood cells.

Column chromatographic, electrophoretic and immunological studies showed the distribution of AMP deaminase isozymes in human blood cells as follows: isozyme E1 in erythrocyte, E2 in granulocyte, L in mononuclear cells, platelets and T-lymphoblast, and probably E1-L hybrid sets in B-lymphoblast.

Platelet AMP deaminase. Regulation by Mg-ATP2- and inorganic phosphate and inhibition by the transition state analog coformycin.

Kinetic studies with platelet AMP deaminase, at pH 7.0 and 100 mM NaC1, gave cooperative initial velocity curves with AMP as substrate, with Mg-ATP2- as an activator, and with Pi as an inhibitor. In the absence of Mg-ATP2-, the s0.5 for AMP was 4.5 mM with a Hill coefficient approaching 2.0. In the presence of saturating Mg-ATP2-, the s0.5 for AMP was reduced to 0.18 mM, the maximum velocity was increased by about 35%, and the Hill coefficient was 1.0. The half-activation constant for Mg-ATP2- varied from 0.7 to 0.07 mM as the concentration of AMP was varied from 0.1 to 5.0 mM and the Hill coefficient for Mg-ATP activation changed from 2.0 to 1.0 over the same range. Phosphate inhibition was competitive with AMP and with Mg-ATP2- (Ki = 2.0 mM) and reversed the activation by Mg-ATP2-. Coformycin inhibited the Mg-ATP-activated enzyme with a Ki less than 0.25 microM. Coformycin inhibition was slow, with a second order rate constant of 6.0 X 10(4) M-1 min-1, suggesting that the compound acts as a transition state analog according to Frieden, C., Kurz, L. C., and Gilbert, H. R. (1980) Biochemistry 19, 5303-5309. The kinetic properties of the enzyme indicate that substantial regulation can occur through changes in AMP concentration acting synergistically to enhance Mg-ATP2- binding and displace Pi from a single type of regulatory site.

AMP deaminase from dogfish erythrocytes: purification and some properties.

The AMP deaminase from erythrocytes from the dogfish, Scyliorhinus caniculus, L., was purified by chromatography on cellulose-phosphate and DEAE-cellulose. The 1300-fold purified enzyme was highly unstable. At low substrate concentrations, a sharp pH optimum was found at pH 6.7. At higher substrate concentrations, the highest activity was found between pH 6.8 and 7.2. The substrate saturation curve was hyperbolic in the presence of monovalent cations. The binding of inorganic phosphate showed cooperativity. Overall the kinetic properties of AMP deaminase from erythrocytes from dogfish are very similar to that described for other species.

Coformycin inhibition of platelet AMP deaminase has no effect on thrombin-induced platelet secretion nor on glycolysis or glycogenolysis.

Thrombin-stimulated platelet secretion is accompanied by a 30% reduction in the steady state level of cytosolic ATP, a breakdown that proceeds through ADP, AMP, IMP, and inosine to hypoxanthine. The ATP to hypoxanthine conversion could be blocked at the stage of AMP deamination by incubation of platelet-rich plasma for 6 h with 200 microM coformycin, a transition-state analog inhibitor of AMP deaminase. Abolition of AMP deaminase activity had no effect on thrombin-induced secretion from the dense granules, alpha-granules, or acid hydrolases measured in gel-filtered platelets. Coformycin treatment had no effect on thrombin-stimulated lactate production, even when oxidative phosphorylation was blocked by antimycin A, nor on the rate of thrombin-stimulated glycogenolysis. In addition, although it was clear that the adenylate energy charge was maintained by activation of AMP deaminase following thrombin treatment, the adenylate energy charge was also maintained in coformycin-treated platelets, albeit after a short lag, by stimulated ATP production and equilibration through the adenylate kinase reaction. Hydrogen peroxide brings about similar adenylate degradation which could also be inhibited by coformycin. The results indicate that AMP deamination and secretion, although temporally related, are not coupled. The role of AMP deaminase appears to be to maintain the adenylate energy charge in the absence of stimulation of ATP production or to buffer the adenylate charge before ATP production is stimulated.

Serum deoxycytidylate deaminase as an index of high-risk pregnancy.

Serum deoxycytidylate deaminase (EC 3.5.4.12, dCMP deaminase) activity was routinely estimated over 7 years in 2460 pregnancies. The results confirm that elevated dCMP deaminase activity (greater than 4.8 units) is associated with the development of pre-eclampsia but not with essential hypertension in pregnancy; a high rising level can precede eclampsia. Elevated enzyme activity was also found in women who developed jaundice in pregnancy and in some with apparently normal pregnancies. An unexpected finding was that these so called 'false positive' high levels were associated with all the unexplained intrauterine fetal deaths that occurred later in pregnancy. Although elevated dCMP deaminase levels may be associated with a normal outcome, persisting high levels indicate the need for careful monitoring of fetal well being and, in the presence of any other sign of placental insufficiency, delivery should be expedited.

Platelet AMP deaminase. Purification and kinetic studies.

AMP deaminase has been purified to homogeneity from human platelets by phosphocellulose chromatography. Kinetic studies showed sigmoidal behavior as a function of AMP concentration with the midpoint of the saturation curve (S0.5) at 3.5 and 4.0 mM in NaCl and KCl, respectively, at pH 6.5. Activation by saturating ATP converted the velocity versus substrate plot to hyperbolic with a Michaelis constant of 1.2 mM and the same maximum velocity in either salt. Addition of increasing concentrations of GTP in the presence of NaCl led to activation followed by inhibition whereas GTP in the presence of KCl gave inhibition with no apparent activation.