Graphene oxide based fluorescent aptasensor for adenosine deaminase detection using adenosine as the substrate

We present a novel fluorescent aptasensor for simple and accurate detection of adenosine deaminase (ADA) activity and inhibition on the basis of graphene oxide (GO) using adenosine (AD) as the substrate. This aptasensor consists of a dye-labeled single-stranded AD specific aptamer, GO and AD. The fluorescence intensity of the dye-labeled AD specific aptamer is quenched very efficiently by GO as a result of strong π-π stacking interaction and excellent electronic transference of GO. In the presence of AD, the fluorescence of the GO-based probe is recovered since the competitive binding of AD and GO with the dye-labeled aptamer prevents the adsorption of dye-labeled aptamer on GO. When ADA was introduced to this GO-based probe solution, the fluorescence of the probe was quenched owing to ADA can convert AD into inosine which has no affinity to the dye-labeled aptamer, thus allowing quantitative investigation of ADA activity. The as-proposed sensor is highly selective and sensitive for the assay of ADA activity with a detection limit of 0.0129U/mL in clean buffer, which is more than one order of magnitude lower than the previous reports. Meanwhile, a good linear relationship with the correlation coefficient of R=0.9922 was obtained by testing 5% human serum containing a series of concentrations of ADA. Additionally, the inhibition effect of erythro-9-(2-hydroxy-3-nonyl) adenine on ADA activity was investigated in this design. The GO-based fluorescence aptasensor not only provides a simple, cost-effective and sensitive platform for the detection of ADA and its inhibitor but also shows great potential in the diagnosis of ADA-relevant diseases and drug development.     

Kinetic characteristics and binding process of substrate analogs to the adenosine deaminase in the marine mussel, Mytilus edulis

1. The purified mussel enzyme deaminated several adenosine analogs with different Km and relative Vmax values. Affinity for adenine was similar to that for adenosine but the deamination rate was extremely slow. 2. Purine riboside was competitive, coformycin was a tight, slow binding inhibitor, and inhibition by both these compounds was pH-dependent. 3. Inosine, hypoxanthine, guanosine and 6-mercapto-purine riboside were slightly inhibitory. 4. The results suggested that initial binding of the substrate was guided by the adenine moiety followed by a stereospecific steering due to a ribose-dependent distortion in the complex to facilitate nucleophilic attack at C-6.     

Studies of nucleosides and nucleotides.: LVIII. Deamination of adenosine analogs with calf intestine adenosine deaminase

Several synthetic adeonosine analogs: 8-fluoro-, 8-azido-, 8-iodo-, 8-methylthioadenosine; 8-bromo-2′-deoxyadenosine, 8-bromoxylofuranosyladenine, 5′-benzoly-8-bromoadenosine; 8,2′-S-, 8,2′-O-, 8,2′-NH-, 8,2′-N-CH₃-, 8,3′,-S-, 8,3′-O-, 8,5′-S- and 8,5′O-cycloadenosine; 1-deaza- and 3-deazaadenosine, as well as tubercidine (7-deazaadenosine), were tested as substrates of calf intestine adenosine deaminase.It was found that the adenine base of adenosine should be in the range φ_rmCN = 0–120° (anti to syn-anti) and 8-fluoroadenosine was hydroylzed very slowly. The purine base should have N¹, N³ or N⁷ atoms for the hydrolysis and only 1-deazaadenosine revealed an inhibitory effect toward the hydrolysis of adenosine.5′-OH group should be in the position of S-configuration and must not be substituted.     

Double-stranded RNA adenosine deaminase ADAR-1-induced hypermutated genomes among inactivated seasonal influenza and live attenuated measles virus vaccines

We sought to examine ADAR-1 editing of measles and influenza virus genomes derived from inactivated seasonal influenza and live attenuated measles virus vaccines grown on chicken cells as the culture substrate. Using highly sensitive 3DI-PCR (R. Suspène et al., Nucleic Acids Res. 36:e72, 2008), it was possible to show that ADAR-1 could hyperdeaminate adenosine residues in both measles virus and influenza virus A genomes. Detailed analysis of the dinucleotide editing context showed preferences for 5'ArA and 5'UrA, which is typical of editing in mammalian cells. The hyperedited mutant frequency, including genomes and antigenomes, was a log greater for influenza virus compared to measles virus, suggesting a greater sensitivity to restriction by ADAR-1.     

Affinity labeling of histidine and lysine residue in the adenosine deaminase substrate binding site.

1. Adenosine deaminase was inactivated by 9-(4-bromoacetamidobenzyl)-adenine (I) and 9-(2-bromoacetamidobenzyl)adenine (II), two affinity labels. 2. The stoichiometry of the reaction with reagent II is reported: 1 mol reagent is bound per mol inactive enzyme. Amino acid analysis of the 6 N HCl hydrolyzate of the inactive enzyme identified CM-histidine as the main alkylation product. This is the first evidence of the presence of a histidine in the active site region. 3. The alkylation rate and involved amino acid residues were studied for both reagents I and II, at pH 8 and 5.5. The particular reactivity of a lysine near or in the active site is discussed.     

Erythrocyte adenosine deaminase in Malaysians.

944 adenosine deaminase phenotypings of Malay, Chinese, and Indian blood donors and newborns at Kuala Lumpur, Malaysia, yielded ADA1 gene frequency estimates of 0.885 for the Malays, 0.939 for the Chinese, and 0.853 for the Indians.     

Human adenosine deaminase. Distribution and properties.

Adenosine deaminase exists in multiple molecular forms in human tissue. One form of the enzyme appears to be "particulate". Three forms of the enzyme are soluble and interconvertible with apparent molecular weights of approximately 36,000, 114,000, and 298,000 (designated small, intermediate, and large, respectively). The small form of adenosine deaminase is convertible to the large form only in the presence of a protein, which has an apparent molecular weight of 200,000 and has no adenosine deaminase activity. This conversion of the small form of the enzyme to the large form occurs at 4 degrees, exhibits a pH optimum of 5.0 to 8.0, and is associated with a loss of conversion activity. The small form of the enzyme predominates in tissue preparations exhibiting the higher enzyme-specific activities and no detectable conversion activity. The large form of adenosine deaminase predominates in tissue extracts exhibiting the lower enzyme specific activities and abundant conversion activity. The small form of adenosine deaminase shows several electrophoretic variants by isoelectric focusing. The electrophoretic heterogeneity observed with the large form of the enzyme is similar to that observed with the small form, with the exception that several additional electrophoretic variants are uniformly identified. No organ specificity is demonstrable for the different electrophoretic forms. The kinetic characteristics of the three soluble molecular species of adenosine deaminase are identical except for pH optimum, which is 5.5 for the intermediate species and 7.0 to 7.4 for the large and small forms.     

A simple rapid fluorescent assay for adenosine deaminase activity.

A simple, rapid (2 hours), fluorescent test for the activity of blood adenosine deaminase (ADA) is described. The test which can be performed on both heparinized and dried blood, is based on the conversion of adenosine to inosine and ammonium in the presence of ADA. The enzyme activity is visually estimated by the oxidation of NADH (fluorescent) to NAD+ (non-fluorescent) in a coupled reaction with glutamate dehydrogenase. The disappearance of fluorescence indicates ADA activity in the sample. The advantages are discussed of the use of this test for the study of the autosomal recessive severe combined immunodeficiency.     

Human adenosine deaminase. Purification and subunit structure.

Human erythrocyte adenosine deaminase has been purified approximately 800,000-fold to apparent homogeneity using antibody affinity chromatography. The enzyme was shown to be a single polypeptide chain with an estimated molecular weight of approximately 38,000. The three electrophoretic forms of erythrocyte adenosine deaminase purified simultaneously by this technique were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Several properties of the highly purified adenosine deaminase including pH optimum, Km for substrate, Ki for product, Stokes radius, sedimentation coefficient, and apparent substrate specificity were identical with the properties observed with an impure preparation of the enzyme.     

ADA2 isoform of adenosine deaminase from pleural fluid

Adenosine deaminase isoenzyme 2 (ADA2) was isolated from human pleural fluid for the first time. Molecular and kinetic properties were characterized. It was shown that the inhibitors of adenosine deaminase isoenzyme 1 (ADA1), adenosine, and erithro-9-(2-hydroxy-3-nonyl)adenine (EHNA) derivatives are poor inhibitors of ADA2. Comparison of the interaction of ADA2 and ADA1 with adenosine and its derivative, 1-deazaadenosine, indicates that the isoenzymes have similar active centers. The absence of ADA2 inhibition by EHNA is evidence of a difference of these active centers in a close environment. The possible role of Zn2+ ions and the participation of acidic amino acids Glu and Asp in adenosine deamination catalyzed by ADA2 were shown.     

Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.

We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.