Partial adenosine deaminase deficiency: another family from southern Africa

Summary Adenosine deaminase (ADA) from a partially ADA-deficient Xhosa man has been characterized. This is only the second such case described in southern Africa, the previous one being a Kalahari San (Bushman). Red blood cell ADA levels were found to be only 6–9% of normal whereas his white cell ADA levels were much higher at 30% of normal. The stability of the enzyme at 57°C was shown to be greatly decreased indicating a mutation resulting in an enzyme with decreased stability in vivo. The Michaelis constant (K_m) for adenosine was found to be normal. Deoxy-ATP levels in the red cells were elevated 2- to 3-times above normal, although this appears to be of no immunological consequence. Starch gel electrophoresis of red cell ADA from family members of the index case, in conjunction with red cell ADA activity levels, suggested that both parents carried a gene for partial ADA deficiency. Isoelectric focusing studies suggested that the two parental partial ADA-deficiency genes were not the same. Electrophoretic studies also revealed that another rare allele of ADA, possibly ADA*5, was segregating within the same family although this event appears to be unconnected with the ADA partial deficiency. A Xhosa population sample was assayed for red cell ADA activity. The results suggested a frequency of 0.015 ± 0.010 for ADA partial-deficiency alleles, although the number of different alleles involved is not known.     

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K_m values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K_i value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.     

Molecular Recognition of Adenosine Deaminase: 15N NMR Studies

The elucidation of the molecular recognition of adenosine deaminase (ADA), the interpretation of the catalytic mechanism, and the design of novel inhibitors are based mostly on data obtained for the crystalline state of the enzyme. To obtain evidence for molecular recognition of the physiologically relevant soluble enzyme, we studied its interactions with the in situ formed inhibitor, 6-OH-purine riboside (HDPR), by 1D-¹⁵N- and 2D-(¹H-¹⁵N)- NMR using the labeled primary inhibitor [¹⁵N₄]-PR. We synthesized both [¹⁵N₄]-PR and an [¹⁵N₄]-HDPR model, from relatively inexpensive ¹⁵N sources. The [¹⁵N₄]-HDPR model was used to simulate H-bonding and possible Zn²⁺-coordination of HDPR with ADA. We also explored possible ionic interactions between PR and ADA by ¹⁵N-NMR monitored pH-titrations of [¹⁵N₄]-PR. Finally, we investigated the [¹⁵N₄]-PR-ADA 1:1 complex by 2D-(¹H-¹⁵N) NMR. We found that HDPR recognition determinants in ADA do not include any ionic-interactions. HDPR N1 H is an H-bond acceptor, and not an H-bond donor. Despite the proximity of N7 to the Zn²⁺-ion, no coordination occurs; instead, N7 is an H-bond acceptor. We found an overall agreement between the crystallographic data for the crystallized ADA:HDPR complex and the ¹⁵N-NMR signals for the corresponding soluble complex. This finding justifies the use of ADA's crystallographic data for the design of novel inhibitors.     

Serum albumin and erythrocyte adenosine deaminase polymorphisms in Asian macaques with special reference to taxonomic relationships amongMacaca assamensis,M. radiata,andM. mulatta

Serum albumin (Alb) and erythrocyte adenosine deaminase (ADA) polymorphisms in Asian macaques were investigated by means of starch gel electrophoresis. The materials comprise a total of 2,323 blood samples from eight species, namely,Macaca fuscata,M. mulatta,M. cyclopis,M. fascicularis,M. nemestrina,M. speciosa,M. radiata, andM. assamensis. It was observed that three Alb phenotypes were controlled by two codominant alleles, Alb _mac ^A and Alb _mac ^B and six ADA phenotypes by four codominant alleles, ADA _mac ¹ , ADA _mac ² , ADA _mac ³ , and ADA _mac ⁴ . The taxonomic relationships amongM. assamensis,M. radiata, andM. mulatta were analyzed by measuring theNei's (1975) genetic distance. The result supportedHill andBernstein's (1969) postulation thatM. assamensis was more closely related phylogenetically toM. radiata than toM. mulatta.     

Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: Implications for modulation of the membrane-associated protein kinase A

In this work, the metabolism of adenosine by isolated BLM associated-enzymes and the implications of this process for the cAMP-signaling pathway are investigated. Inosine was identified as the major metabolic product, suggesting the presence of adenosine deaminase (ADA) activity in the BLM. This was confirmed by immunoblotting and ADA-specific enzyme assay. Implications for the enzymatic deamination of adenosine on the receptor-modulated cAMP-signaling pathway were also investigated. We observed that inosine induced a 2-fold increase in [³⁵S] GTPγS binding to the BLM and it was inhibited by 10⁻⁶ M DPCPX, an A₁ receptor-selective antagonist. Inosine (10⁻⁷ M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and G_αi-3 protein-coupled A₁ receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A₁ receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A₁ receptor-mediated inhibition of adenylyl cyclase.     

Erythrocyte acid phosphatase (ACP1) activity

Summary Erythrocyte acid phosphatase (ACP₁) activity was determined in the absence of modulators and in the presence of either adenosine or inosine as modulators in 154 samples of red blood cells collected from adult donors. Adenosine and inosine showed modulating effects (activation), that were genotype dependent in the allele order p^bac; the activation by inosine was much higher than by adenosine. The modulating effect was dependent on adenosine deaminase (ADA) genotype: In carriers of ADA² allele the activation with ACP₁ phenotype A was lower and that with phenotypes CA and CB was higher than in ADA¹/ADA¹ subjects. In addition, the basic ACP₁ activity (i.e., without modulators) also appeared to be dependent on ADA genotype: The lowest ACP₁ activity was observed in A and BA subjects carrying the ADA² allele. Since the deamination of adenosine to inosine associated with ADA2-1 phenotype is slower than that associated with ADA1, the interaction of ADA on ACP₁ activity may in fact be explained by a lower intracellular concentration of inosine in ADA² carriers and, therefore, by a lower modulating effect of this on acid phosphatase activity.     

Free metal ion depletion by "Good's" buffers. I. N-(2-acetamido)iminodiacetic acid 1:1 complexes with calcium(ii). magnesium(II), zinc(II), manganese(II), cobalt(II), nickel(II), and copper(II).

Formation (affinity) constants for 1:1 complexes of N-(2-acetamido)iminodiacetic acid (ADAH₂) with Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) have been determined. Probable structures of the various metal chelates existing in solution are discussed. Values for the deprotonation of the amide group in [Cu(ADA)] and subsequent hydroxo complex formation are also reported. The use of ADA as a buffer is considered in terms of metal buffers complexes which can be formed at physiological pH, i.e., at pH 7.0 there is essentially no free metal ion in 1:1 M²⁺ to ADA solutions.     

The effect of G2-treatments with 2′-deoxyadenosine on the frequency of chromatid aberrations in human lymphocytes depends on the type of culture

The effect of G₂-treatments with 2-deoxyadenosine (dAdo) on the frequency of chromatid aberrations in X-irradiated and unirradiated human lymphocytes depends on the method of culture. In whole-blood cultures dAdo alone produced very few if any aberrations, but in the presence of inhibitors of adenosine deaminase (ADA), such as EHNA or coformycin, a high frequency of chromatid gaps, chromatid breaks, and isochromatid breaks were produced. In cultures of purified lymphocytes, dAdo produced aberrations even in the absence of an ADA inhibitor. Apparently the lymphocytes are protected against the chromosome-damaging effect of dAdo by the ADA activity of the erythrocytes. — When given as a post-treatment, dAdo also enhances the frequency of chromatid aberrations induced by X-rays in G₂. In whole-blood cultures this effect is obtained even in the absence of an ADA inhibitor, although the concentration required to produce enhancement is about twenty times higher than in the presence of the inhibitor.     

An in vitro comparison of a new vinyl chalcogenide and sodium selenate on adenosine deaminase activity of human leukocytes

Selenium (Se) is a dietary essential trace element with important biological roles. Sodium selenate (Na₂SeO₄) is an inorganic Se compound used in human and animal nutrition that acts as precursor for selenoprotein synthesis. The organoselenium 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one (C₂₁H₂HOSe) is an α,β-unsaturated ketone functionalized vinyl chalcogenide that has been found as a potential tool in organic synthesis. Adenosine deaminase (ADA) is an important enzyme in the degradation of adenine nucleotides. In this study, we investigated the in vitro effects of both Se compounds on ADA activity and cell viability in leukocyte suspension (LS) of healthy donors (n = 12). We first observed an inhibition of ADA activity using 0.1 μM of 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one, and an increase in cellular viability when 30 μM were used. However, we did not observe alterations in the presence of sodium selenate. Moreover, both Se compounds did not alter lactate dehydrogenase activity and thiobarbituric acid reactive substance levels. These results suggest that the inhibition of ADA activity caused by α,β-unsaturated ketone may affect the adenosine levels in LS and modulate cell viability, attenuating conditions that involve the activation of the immune system.     

Altering drug tolerance of surface plasmon resonance assays for the detection of anti-drug antibodies

Anti-drug antibody (ADA) responses are a concern for both drug efficacy and safety, and high drug concentrations in patient samples may inhibit ADA assays. We evaluated strategies to improve drug tolerance of surface plasmon resonance (SPR) assays that detect ADAs against a bispecific Adnectin drug molecule that consists of an anti-VEGFR2 domain linked to an anti-IGF-1R domain (V-I-Adnectin). Samples containing ADAs against V-I-Adnectin and various drug concentrations were tested in the presence of 1 M guanidine hydrochloride (Gdn), at pH values ranging from 4.5 to 7.4 and temperatures of up to 37 °C. Temperature had a negligible effect in weakening the affinity of interaction of monoclonal antibodies with polyethylene glycol(PEG)–V-I-Adnectin and did not increase drug tolerance of the ADA assay. Low pH increased drug tolerance of the assay relative to pH 7.4 but caused nonspecific binding of the drug during competition experiments. The chaotropic agent Gdn lowered the affinity of interaction between an anti-V-Adnectin monoclonal antibody and the drug (from K_D = 0.93 nM to K_D = 348 nM). That decrease in the affinity of drug–ADA interaction correlated with an increase of assay drug tolerance. Conditions that lower drug–ADA interaction affinity could also be used to develop drug-tolerant SPR assays for other systems.     

Evidence of selective interaction between adenosine deaminase and acid phosphatase polymorphisms in fetuses carried by diabetic women

Summary Possible selective interaction between genetic polymorphisms of acid phosphatase locus 1 (ACP₁) and adenosine deaminase (ADA) has been investigated in a sample of 211 infants from diabetic women, and in 350 consecutive infants from normal women. Newborns from diabetic pregnancies carrying the ADA² allele show a lower proportion of BA and CB phenotypes (heterozygotes for the main allele of ACP₁ system), compared with both their mothers and normal infants. The observation suggests that, in a diabetic environment, intrauterine selection may act against double heterozygotes for the ACP₁ and ADA systems.     

Trypanosoma evansi: Adenosine deaminase activity in the brain of infected rats

The study was undertaken to evaluate changes in the activity of adenosine deaminase (ADA) in brains of rats infected by Trypanosoma evansi. Each rat was intraperitoneally infected with 10⁶ trypomastigotes either suspended in fresh (group A; n = 13) and cryopreserved blood (group B; n = 13). Thirteen animals were used as control (group C). ADA activity was estimated in the cerebellum, cerebral cortex, striatum and hippocampus. No differences (P > 0.05) in ADA activity were observed in the cerebellum between infected and non-infected animals. Significant (P < 0.05) reductions in ADA activity occurred in cerebral cortex in acutely (day 4 post-infection; PI) and chronically (day 20 PI) infected rats. ADA activity was significantly (P < 0.05) decreased in the hippocampus in acutely infected rats, but significantly (P < 0.05) increased in the chronically infected rats. Significant (P < 0.05) reductions in ADA activity occurred in the striatum of chronically infected rats. Parasites could be found in peripheral blood and brain tissue through microscopic examination and PCR assay, respectively, in acutely and chronically infected rats. The reduction of ADA activity in the brain was associated with high levels of parasitemia and anemia in acute infections. Alterations in ADA activity of the brain in T. evansi-infected rats may have implications for pathogenesis of the disease.     

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH₃, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples.     

Interaction at clinical level between erythrocyte acid phosphatase and adenosine deaminase genetic polymorphisms

Summary The effects of ACP₁ phenotype on birth weight, neonatal jaundice, and obesity in children are dependent on ADA genotype. These phenomena may represent a clinical counterpart of the in vitro biochemical interactions between the two systems recently observed by our group.     

A study on the inhibition of adenosine deaminase

Adenosine deaminase (ADA, EC 3.5.4.4) catalyses the irreversible deamination of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively. In this study the inhibition of ADA from bovine spleen by several molecules with structure related to that of the substrate or product has been quantified. The inhibitors adenine, purine, inosine, 2-aminopurine, 4-aminopyrimidine, 4-aminopyridine, 4-hydroxypyridine and phenylhydrazine are shown to be competitive inhibitors with K_I (mM) values of 0.17, 1.1, 0.35, 0.33, 1.3, 1.8, 1.4 and 0.25, respectively. Synergistic inhibition by various combinations of molecules that imitate the structure of the substrate has never been observed. Some general conclusions are: i) the enzyme ADA from bovine spleen we have used is appropriate for kinetic studies of inhibition and mechanistic studies; it can be a reference catalytic system for the homogeneous comparison of various inhibitors; ii) this enzyme presents very rigid requirements for binding the substrate: variations in the structure of adenosine imply the loss of important interactions.     

Erythrocytenenzyme der Primaten

Zusammenfassung Die Adenosindesaminasen der Primaten zeigen eine genetisch determinierte Variabilität. Bei der Untersuchung von 327 subhumanen Primaten (289 Simiae der Alten Welt, 38 Prosimiae) konnten wir neun Adenosindesaminase-Varianten nachweisen, die auf Grund ihrer unterschiedlichen elektrophoretischen Wandergeschwindigkeit als ADA 6, ADA 4, ADA 2, ADA 2, ADA 1, ADA 3, ADA 5, ADA 5 und ADA 7 bezeichnet werden. Die Verteilung der verschiedenen Phänotypen wurde ermittelt.

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Red cell enzymes of primatesAdenosine deamisnase (EC: 3.5.4.4.)

Summary The polymorphism of adenosine deaminase has been investigated in 327 subhuman Primates (289 Old World Monkeys and 38 Prosimians). Nine adenosine deaminase variants were found to be present, which on the basis of their different electrophoretic mobilities were designated ADA 6, ADA 4, ADA 2, ADA 2, ADA 1, ADA 3, ADA 5, ADA 5 and ADA 7. The distribution of these various phenotypes has been estimated.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Adenosine deaminase 2 from chicken liver: purification, characterization, and N-terminal amino acid sequence

Adenosine deaminase (ADA) is involved in purine metabolism and plays an important role in the mechanism of the immune system. ADA activity is composed of two kinetically distinct isozymes, which are referred to as ADA1 and ADA2. ADA1 is widely distributed in many animals and well characterized. On the contrary, relatively little is known about ADA2. In this study, we first purified ADA2 to homogeneity from chicken liver. The purified enzyme had a molecular mass of approximately 110 kDa on gel filtration. Also, the enzyme was shown to be a homodimer with an estimated molecular mass of 61 kDa on SDS-PAGE. Following treatment with N-glycosidase, the molecular mass of ADA2 changed to 55 kDa. Several properties of the highly purified ADA2 were also investigated in this study. Furthermore, the N-terminal amino acid sequence of ADA2 was determined.     

Adamantanes Enhance the Photovoltaic Performance and Operational Stability of Perovskite Solar Cells by Effective Mitigation of Interfacial Defect States

Passivation of electronic defects is an effective strategy to boost the performance and operational stability of perovskite solar cells (PSCs). Identifying molecular materials that achieve this purpose is a key target of current research efforts. Here, adamantane (AD) and 1‐adamantylamine (ADA) are introduced as molecular modulators to abate electronic defects present within the bulk and at the perovskite–hole conductor interface. To this effect, the modulator is added either into the antisolvent (AS) to precipitate it together with the perovskite (AS method) or they are spin coated (SC) onto its surface (SC method). Time‐resolved photoluminescence measurements show substantially longer lifetimes for perovskite films treated with AD and ADA compared to the reference sample. In line with this observation, it is found that the presence of AD and ADA molecules at the interface between the perovskite film and the hole conductor increases all photovoltaic metrics, in particular the open circuit photovoltage (V _oc) as well as the operational stability of the PSC.