Cell surface adenosine deaminase binds and stimulates plasminogen activation on 1-LN human prostate cancer cells

Adenosine deaminase (ADA) is expressed intracellularly by all cells, but in some tissues, it is also associated with the cell surface multifunctional glycoprotein CD26/dipeptidyl peptidase IV. By modulating extracellular adenosine, this "ecto-ADA" may regulate adenosine receptor signaling implicated in various cellular functions. CD26 is expressed on the surface of human prostate cancer 1-LN cells acting as a receptor for plasminogen (Pg). Since ADA and Pg bind to CD26 at distinct but nearby sites, we investigated a possible interaction between these two proteins on the surface of 1-LN cells. Human ADA binds to CD26 on the surface 1-LN cells and immobilized CD26 isolated from the same cells with similar affinity. In both cases, ADA binding is diminished by mutation of ADA residues known to interact with CD26. ADA was also found to bind Pg 2 in the absence of CD26 via the Pg kringle 4 (K4) domain. In the presence of 1-LN cells or immobilized CD26, exogenous ADA enhances conversion of Pg 2 to plasmin by 1-LN endogenous urinary plasminogen activator (u-PA), as well as by added tissue Pg Activator (t-PA), suggesting that ADA and Pg bind simultaneously to CD26 in a ternary complex that stimulates the Pg activation by its physiologic activators. Consistent with this, in melanoma A375 cells that bind Pg, but do not express CD26, the rate of Pg activation was not affected by ADA. Thus, ADA may be a factor regulating events in prostate cancer cells that occur when Pg binds to the cell surface and is activated.     

CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa.

Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, which showed the localization of CD26 at the postacrosomal region and at the midpiece of the spermatozoa after incubation with vesicles. Moreover, a similar localization of CD26 and ecto-ADA on the spermatozoa was evidenced after fusion. Our results suggest that the interaction CD26/ecto-ADA might be responsible for fusion. Since A(1)ARs are said to be second receptors for ecto-ADA to form ecto-ADA/A(1)AR complexes, and since horse spermatozoa have A(1)ARs at their surface, the interaction CD26/ecto-ADA/A(1)AR during the fusion process cannot be ruled out.     

The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane

A(1) adenosine receptors (A(1)Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A(1)Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 +/- 0.1 nM). The interaction between hsc73 and A(1)R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from (35)S-labeled guanosine-5'-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A(1)Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A(1)R and hsc73 was detected in DDT(1)MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A(1)R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A(1)Rs by two qualitatively different vesicle types, one in which A(1)R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins.     

Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A₁ adenosine receptor (A₁R) is a second ecto-ADA binding protein. Binding of ADA to A₁R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A₁R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via A₁R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via A₁R.     

Selective inactivation or reconstitution of adenosine A2A receptors in bone marrow cells reveals their significant contribution to the development of ischemic brain injury

Inactivation of the adenosine A(2A) receptor (A(2A)R) consistently protects against ischemic brain injury and other neural insults, but the relative contribution of A(2A)Rs on peripheral inflammatory cells versus A(2A)Rs expressed on neurons and glia is unknown. We created a chimeric mouse model in which A(2A)Rs on bone marrow-derived cells (BMDCs) were selectively inactivated or reconstituted by bone marrow transplantation. Selective reconstitution of A(2A)Rs on BMDCs (A(2A)R knockout mice transplanted with wild-type bone marrow cells) largely reinstates ischemic brain injury in global A(2A)R knockout mice. Conversely, selective inactivation of A(2A)Rs on BMDCs (wild-type mice transplanted with A(2A)R knockout bone marrow cells) attenuates infarct volumes and ischemia-induced expression of several proinflammatory cytokines in the brain, but exacerbates ischemic liver injury. These results indicate that the A(2A)R-stimulated cascade in BMDCs is an important modulator of ischemic brain injury and that ischemic brain and liver injuries are regulated distinctly by A(2A)Rs on BMDCs.     

Clustered charged amino acids of human adenosine deaminase comprise a functional epitope for binding the adenosine deaminase complexing protein CD26/dipeptidyl peptidase IV

Human adenosine deaminase (ADA) occurs as a 41-kDa soluble monomer in all cells. On epithelia and lymphoid cells of humans, but not mice, ADA also occurs bound to the membrane glycoprotein CD26/dipeptidyl peptidase IV. This "ecto-ADA" has been postulated to regulate extracellular Ado levels, and also the function of CD26 as a co-stimulator of activated T cells. The CD26-binding site of human ADA has been localized by homolog scanning to the peripheral alpha2-helix (amino acids 126-143). Among the 5 non-conserved residues within this segment, Arg-142 in human and Gln-142 in mouse ADA largely determined the capacity for stable binding to CD26 (Richard, E., Arredondo-Vega, F. X., Santisteban, I., Kelly, S. J., Patel, D. D., and Hershfield, M. S. (2000) J. Exp. Med. 192, 1223-1235). We have now mutagenized conserved alpha2-helix residues in human and mouse ADA and used surface plasmon resonance to evaluate binding kinetics to immobilized rabbit CD26. In addition to Arg-142, we found that Glu-139 and Asp-143 of human ADA are also important for CD26 binding. Mutating these residues to alanine increased dissociation rates 6-11-fold and the apparent dissociation constant K(D) for wild type human ADA from 17 to 112-160 nm, changing binding free energy by 1.1-1.3 kcal/mol. This cluster of 3 charged residues appears to be a "functional epitope" that accounts for about half of the difference between human and mouse ADA in free energy of binding to CD26.     

Human adenosine deaminase as an allosteric modulator of human A(1) adenosine receptor: abolishment of negative cooperativity for [H](R)-pia binding to the caudate nucleus

It has been shown that adenosine deaminase (ADA; EC 3.5.4.4) behaves as an ecto-enzyme anchored to membrane proteins, among them A(1) adenosine receptors (A(1)Rs). Bovine ADA interacts with A(1)Rs from many species and regulates agonists binding to receptors in an activity-independent form. However, it was not known whether human ADA exerted any effect on the agonist binding to human A(1)Rs, because of both technical difficulties in obtaining pure human ADA and tissues containing human A(1)Rs. In this study, human ADA was purified to homogeneity. Taking in consideration that A(1)Rs form homodimers and taking advantage of a new procedure to fit binding data to receptors dimers, which allows to calculate ligand dissociation constants and the degree of cooperativity between the two subunits in the dimer, here it is demonstrated that human ADA markedly enhances the agonist and antagonist affinity and abolishes the negative cooperativity on agonist binding to human striatal A(1)Rs. ADA also increases the ability of the agonist to decrease the forskolin-induced cAMP levels. The results show that human ADA, apart from reducing the adenosine concentration and thus preventing A(1)R desensitization, binds to A(1)R behaving as an allosteric effector that markedly enhances agonist affinity and increases receptor functionality. The physiological role of the interaction is to make receptors more sensitive to adenosine. This powerful regulation has important implications for the physiology and pharmacology of neuronal A(1)Rs.     

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain and pituitary of the goldfish

Summary The distribution of neuropeptide Y (NPY) immunoreactivity has been studied by means of immunocytochemistry and radioimmunoassay in the brain of the goldfish. It was found that NPY had a widespread distribution in the entire brain in particular in the telencephalon, diencephalon, optic tectum and rhombencephalon. In the pituitary gland, positive type-B fibers were observed in the various lobes frequently in direct contact with secretory cells, in particular the gonadotrophs, somatotrophs and MSH (melanocyte-stimulating hormone) secreting cells. When measured by radioimmunoassay, the highest NPY concentrations were found in the pituitary and telencephalon, confirming the results of immunocytochemistry. The displacement curves obtained with serial dilutions of brain extracts were parallel to that of synthetic porcine NPY. Following high performance liquid chromatography, the NPY-like material extracted from goldfish brain co-eluted as a single peak with synthetic porcine NPY. These data demonstrate the presence of an NPY-like substance widely distributed in the goldfish brain. The observation of NPY-immunoreactive fibers in the pituitary gland suggests that, among its other functions, NPY may play a role in the neuroendocrine regulation of pituitary function.     

Molecular and cellular physiology of aromatase in the brain and retina

Due to exceptionally high brain aromatase activity, teleost fish are advantageous for studying neural aromatase regulation, localization, and physiology. To determine the molecular mechanism of enhanced expression, we have isolated, cloned and sequenced a 3 kb full-length aromatase cDNA from a goldfish (Carassius auratus) brain library using a human placental aromatase cDNA as probe. The deduced sequence of goldfish aromatase is 510 amino acids (predicted M_W, 58 kDa) with 69% overall sequence similarity, when compared to human placental aromatase, and higher hormologies in presumptive functional domains. A major 3 kb mRNA species was abundant in brain and low or non-detectable in non-neural tissues, reflecting the order of enzyme activities. To determine the cellular basis of high enzyme activity in goldfish brain, a human placental aromatase antibody was used to immunolocalize labeled cells. This antibody immunoprecipitated a single 56 kDa in vitro translation product of goldfish brain poly(A⁺)RNA and revealed discrete clusters of intensely stained neurons, processes, and terminals concentrated in, but not limited to, reproductive brain centers. Close proximity of aromatase- and androgen receptor-positive neurons in certain regions provides anatomic evidence of a functional relationship between direct and indirect pathways of neural androgen action. Aromatase-positive neurons and fibers formed interconnected networks in novel loci (e.g. retina→optic tract→optic tectum), and catalytic activity was confirmed biochemically in these tissues, indicating that neuroestrogen may have a role in visual input and integration. Availability of goldfish-specific nucleotide and antibody probes will facilitate further studies using this model.     

Ultrastructural localization of acetylcholinesterase in retino-deprived optic tectum of the goldfish

The ultrastructural localization of AChE has been studied in the optic tectum of the goldfish after unilateral eye ablation. 1 or 4 months after the operation the patterns of enzyme localization were essentially the same in the normal and affected optic tectum, despite structural modifications caused by the degeneration of retinal terminals and dendritic atrophy of some tectal neurons. The results are discussed in relation to the different hypotheses put forward concerning possible cholinergic mechanisms in the optic tectum of teleosts.     

The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ecto-enzymes ecto-5′-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamole-sensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and α-smooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5′-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by high-performance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5′-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5′-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5′-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.     

Goldfish Brain (Na+, K+)-ATPase: Purification of the Catalytic Polypeptide and Production of Specific Antibodies

Abstract: The denatured catalytic polypeptide of (Na⁺, K⁺)-ATPase of goldfish brain was purified and identified as the ³²P-labeled phosphoprotein. The protein served as immunogen for the preparation of rabbit antisera for immunohistochemical application to goldfish tissue sections, using the peroxidase-antiperoxidase indirect method. Labeling in brain cross-sections appears primarily in fibers of the optic nerve layer of the tectum. In optic nerve cross sections, labeling is restricted to fiber bundles.     

Adenosine A2A receptors and metabotropic glutamate 5 receptors are co-localized and functionally interact in the hippocampus: a possible key mechanism in the modulation of N-methyl-D-aspartate effects

Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA-mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A(2A)Rs) modulated mGlu5R-dependent effects in the hippocampus, as they do in the striatum. Co-application of the A(2A)R agonist CGS 21680 with the mGlu5R agonist (RS)-2-chloro-s-hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A(2A)Rs seemed to be required to enable mGlu5R-mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A(2A)R antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A(2A)R knockout mice. Evidence for the interaction between A(2A)Rs and mGlu5Rs was further strengthened by demonstrating their co-localization in hippocampal synapses. This is the first evidence showing that hippocampal A(2A)Rs and mGlu5Rs are co-located and act synergistically, and that A(2A)Rs play a permissive role in mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus.     

Retinal axons inXenopus laevis recognise differences between tectal and diencephalic glial cells in vitro

Explants of retina from Xenopus laevis were cultured on monolayers of tectal and diencephalic glial cells in order to determine whether the glia, normally encountered by optic nerve fibres as they grow to the optic tectum, can influence the growth of these neurons in any way. Explants of nasal retina produced prolific radial outgrowth patterns on both tectal and diencephalic monolayers. Explants of temporal retina produced similar outgrowth patterns on diencephalic glia, but on tectal glia the outgrowth was restricted and fibres were fasciculated in short, fat bundles.     

The antidromic activation of tectal neurons by electrical stimuli applied to the caudal medulla oblongata in the toad,Bufo bufo L.

In order to specify the tectal projection to the bulbar/spinal regions, the antidromic responses of the physiologically identified tectal neurons as well as the gross antidromic field responses in the optic tectum to electrical stimuli applied to the caudal medulla were examined in the paralyzed common toad, Bufo bufo. The antidromic field potential was recorded in the optic tectum in response to electrical stimuli applied to the ventral paramedian portion of the contralateral caudal medulla (where the crossed tecto-spinal pathway of Rubinson (1968) and Lázár (1969) runs), but generally not when they were applied to various parts of the ipsilateral caudal medulla. The antidromic field potential was largest at the superficial part of Layer 6 or at the border between Layers 6 and 7 of the optic tectum, indicating that neurons in these layers project to the contralateral caudal medulla. Mapping experiments of the antidromic field potential over the optic tectum showed that the antidromic field potential was recorded mainly in the lateral part of it, indicating that this part of the optic tectum is the main source of projection neurons to the contralateral caudal medulla. Various classes of tectal neurons as well as retinal ganglion neurons were identified from the characteristics of the response properties to moving visual stimuli and the properties of the receptive fields. Of these, the Class T1, T2, T3, T4, T5(1), T5(2), T5(3), and T5(4) tectal neurons were activated antidromically by stimuli applied to the contralateral caudal medulla. Only a limited proportion of the Class T5(1) neurons was activated antidromically by stimuli applied to the ipsilateral caudal medulla. On the other hand, the Class T7 and T8 neurons, as well as the Class R2, R3, and R4 retinal neurons, were not activated antidromically by stimuli applied to the caudal medulla of either side. These results suggest a possibility that these tectal neurons which project to the medullary regions form the substrate of the sensorimotor interfacing and contribute to the initiation or coordination of the visually guided behavior, such as prey-catching.     

Molecular mechanisms involved in the adenosine A and A receptor-induced neuronal differentiation in neuroblastoma cells and striatal primary cultures

Adenosine A1 receptors (A1Rs) and adenosine A(2A) receptors (A(2A)Rs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A(2A)Rs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A(2A)Rs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A(2A)R-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A(2A)R agonists but not by A1R agonists. PKA activation is therefore necessary for A(2A)R-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A(2A) receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.     

Reparative neurogenesis in the brain and changes in the optic nerve of adult trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> after mechanical damage of the eye

Reparative proliferation and neurogenesis in the brain integrative centers after mechanical eye injury in an adult trout Oncorhynchus mykiss have been studied. We have found that proliferation and neurogenesis in proliferative brain regions, the cerebellum, and the optic tectum were significantly enhanced after the eye injury. The cerebellum showed a significant increase in the proliferative activity of the cells of the dorsal proliferative zone and parenchymal cells of the molecular and granular layers. One week after the injury, PCNA-positive radial glia cells have been identified in the tectum. We have found for the first time that the eye trauma resulted in the development of local clusters of undifferentiated cells forming so called neurogenic niches in the tectum and cerebellum. The differentiation of neuronal cells detected by labeling cells with antibodies against the protein HuC/D occurred in the proliferative zones of the telencephalon, the optic tectum, cerebellum, and medulla of a trout within 2 days after the injury. We have shown that the HuC/D expression is higher in the proliferative brain regions than in the definitive neurons of a trout. In addition, we have examined cell proliferation, migration, and apoptosis caused by the eye injury in the contra- and ipsilateral optic nerves and adjacent muscle fibers 2 days after the trauma. The qualitative and quantitative assessment of proliferation and apoptosis in the cells of the optic nerve of a trout has been made using antibodies against PCNA and the TUNEL method.     

O-GlcNAc modification of radial glial vimentin filaments in the developing chick brain

We examined the post-translational modification of intracellular proteins by β-O-linked N-acetylglucosamine (O-GlcNAc) with regard to neurofilament phosphorylation in the developing chick optic tectum. A regulated developmental pattern of O-GlcNAcylation was discovered in the developing brain. Most notably, discernible staining occurs along radial glial filaments but not along neuronal filaments in vivo. Immunohistochemical analyses in sections of progressive stages of development suggest upregulation of O-GlcNAc in the ependyma, tectofugal neuron bodies, and radial glial processes, but not in axons. In contrast, double-label immunostaining of monolayer cultures made from dissociated embryonic day (E) 7 optic tecta revealed O-GlcNAcylation of most axons. Labeling of brain sections together with Western blot analyses showed O-GlcNAc modification of a few discrete proteins throughout development, and suggested vimentin as the protein in radial glia. Immunoprecipitation of vimentin from E9 whole brain lysates confirmed O-GlcNAcylation of vimentin in development. These results indicate a regulated pattern of O-GlcNAc modification of vimentin filaments, which in turn suggests a role for O-GlcNAc-modified intermediate filaments in radial glia, but not in neurons during brain development. The control mechanisms that regulate this pattern in vivo, however, are disrupted when cells are placed in vitro.     

Neuron-specific enolase-like immunoreactivity in the vertebrate retina: selective labelling of Müller cells in Anura.

Neuron-specific enolase (NSE) immunocytochemistry was carried out in retinae of goldfish, axolotl, clawed frog, cane toad, lizard, chick, guinea-pig, rabbit, rat, cat and human. With the exception of Anura, strong immunoreactivity was seen in the large ganglion, amacrine cells and horizontal cells of the retina in all of the other species. Photoreceptors were found to be labelled in the rat and human retina and only one cone type in rabbit. Photoreceptor pedicles and ellipsoids were stained in the goldfish and the somata and inner segments of some photoreceptors in axolotl. In the axolotl retina, besides neurons, Müller cells (MCs) were also immunolabelled. In the retina of the cane toad and the clawed frog MCs were the only stained elements. Similarly in other parts of the central nervous system of the cane toad, glial elements of the optic tectum and spinal cord were immunoreactive. In contrast, in the peripheral nervous system, neurons of the 1st sympathetic ganglion and the 2nd dorsal root ganglion were labelled. In double-labelling experiments, glial fibrillary acidic protein and NSE showed colocalisation both in the glial elements of the optic tectum and spinal cord and in MCs of the retina of the cane toad.     

Characterization and purification of adenosine deaminase 1 from human and chicken liver

Adenosine deaminase 1 (ADA1) was purified from human and chicken liver. The purified enzyme had a molecular weight of approximately 42,000 Da on SDS-PAGE. In humans, ADA1 was mainly purified concomitant with ADA-binding protein, dipeptidyl peptidase IV (DPP IV)/CD26; however, in chickens, only ADA1 without DPP IV was purified. Both human and chicken ADA1s showed similar properties on substrate specificities, sensitivities on inhibitors, and pH profile. However, they had different affinities with adenosine-Sepharose and IgG anti-ADA1-Sepharose. Human ADA1 was not adsorbed in adenosine-Sepharose column, but chicken ADA1 was adsorbed. As for IgG anti-ADA1-Sepharose column, the results were converse. Furthermore, human ADA1 could bind to DPP IV whereas chicken ADA1 could not.