Association of adenosine deaminase with erythrocyte and platelet plasma membrane: an immunological study using light and electron microscopy

Adenosine deaminase has been localized in the plasma membrane of erythrocytes and platelets by means of immunological techniques using light and electron microscopy with cells in suspension. In erythrocytes, adenosine deaminase is associated with the external side of the plasma membrane. In platelets, the enzyme is associated with the external side of the plasma membrane, which is known to extend through the canalicular system of these cells. These results confirm our previous findings, based on biochemical studies, concerning the attachment of the enzyme to cell membranes.     

Immunomorphological localization of adenosine deaminase in rat tissues during ontogeny

Summary Immunomorphological methods were used to localize adenosine deaminase in tissues of the rat at different stages of ontogeny. In the thymus, lymphocytes began to express significant amounts of the enzyme with the appearance of demarcation between the cortex and medulla at 17 days of gestation. At any stage of ontogeny studied, strong adenosine deaminase staining was seen predominantly in cortical thymocytes. In the spleen and lymph node, the enzyme was initially detected in T cell areas, whereas primary follicles did not show positive adenosine deaminase staining. During further development, the enzyme was demonstrated in some lymphocytes of germinal centres and plasma cells. In the duodenum, epithelial cells of villi and the neck of crypts showed positive adenosine deaminase staining whereas no staining for the enzyme was observed in the epithelial cells of the base of crypts. Strongly positive staining for adenosine deaminase appeared in plasma cells of the lamina propria by four weeks after birth. The transient positive reaction for the deaminase could be recognized in epithelial cells of tubules of the kidney during late foetal and early postnatal development. The tubules of adult rats did not stain for the enzyme. In the cartilage of 15-day foetuses, positive adenosine deaminase staining was seen only in perichondrial cells and hypertrophic cells. Kuppfer cells in the liver and endothelial cells of blood vessels stained positively for the enzyme at every stage of ontogeny studied.     

Adenosine Deaminase Interacts with A1 Adenosine Receptors in Pig Brain Cortical Membranes

Abstract: Adenosine deaminase is an enzyme of purine metabolism that has largely been considered to be cytosolic. A few years ago, adenosine deaminase was reported to appear on the surface of cells. Recently, it has been demonstrated that adenosine deaminase interacts with a type II membrane protein known as either CD26 or dipeptidylpeptidase IV. In this study, by immunoprecipitation and affinity chromatography it is shown that adenosine deaminase and A₁ adenosine receptors interact in pig brain cortical membranes. This is the first report in brain demonstrating an interaction between a degradative ectoenzyme and the receptor whose ligand is the enzyme substrate. By means of this interaction adenosine deaminase leads to the appearance of the high-affinity site of the receptor, which corresponds to the receptor-G protein complex. Thus, it seems that adenosine deaminase is necessary for coupling A₁ adenosine receptors to heterotrimeric G proteins.     

Lymphocyte plasma membranes II. Cytochemical localization of 5′-nucleotidase in rat lymphocytes

Cytochemical studies of thymic and splenic lymphocytes from rats showed that 5′-nucleotidase was restricted to the plasma membranes. Isolated plasma membranes contained the highest specific activity of 5′-nucleotidase of any cellular fractions. The results indicate that this enzyme can be used as a plasma membrane marker for lymphocytes.     

Evaluation of adenosine deaminase assay for analyzing t-lymphocyte density in vitro

Summary The proliferative capacity of T cells in response to various stimuli is commonly determined by radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly synthesized DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, an alternative method was tested. As an alternative, T-cell proliferation was measured by spectrophotometrically analyzing the presence of an enzyme adenosine deaminase in lymphocytes and also using a standard XTT assay. Jurkat (human) T-cell line (clone E6.1) was used for lymphocyte population. The Jurkat cell concentration was adjusted according to different cell densities and enzyme activity was determined. Cells were also seeded in complete medium up to 72 h and harvested for estimation of enzyme activity. A significant correlation between the standard cell-proliferation assay and adenosine deaminase assay was observed. The present study indicates that the assay of adenosine deaminase is a reliable and accurate method for measuring proliferation of T lymphocytes.     

Purification and some properties of the soluble and membrane-bound adenosine deaminases of Micrococcus sodonensis ATCC 11880 and their distribution within the family Micrococcacea.

In Micrococcus sodonensis and some other Micrococcus species, adenosien deaminase is present both as a membran-bound and a soluble enzyme; The membran-bound adenosine deaminase can be extracted with n-butanol, and may account for up to 5% of the total cellular adenosine deaminase activity. In a number oc comparative tests, no differences between the two enzyme forms could be found, thus they are believed to be similar molecular species; The purified membran-bound or soluble enzyme had a molecular weight, obtained by gel-filtration, of 130 000 and was inactive toward adenine and adenine mononucleotides. It appears, therefore, to be more closely related to the calf-intestine enzyme than the Aspergillus oryzae form in respect to size and substrate specificity; Attempts to correlate membrane-bound adenosine deaminase activity with adenosine transport in isolated membrane vesicles of M. sodonensis indicated no obvious relationship between the two activities.     

Inhibition of T lymphocyte activation by cyclosporin A: interference with the early activation of plasma membrane phospholipid metabolism

Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (Con A). Cyclosporin A (CSA) inhibited in a dose-dependent way the induction of RNA and DNA synthesis; nearly complete inhibition was observed at a concentration of 200 ng/ml. Results of kinetic studies suggested that the immunosuppressive drug interfered with an early event occurring in activated lymphocytes. Among the earliest changes detectable in activated lymphocytes, the turnover of plasma membrane phospholipids is increased, predominantly of their fatty acid moieties, catalyzed by the membrane-bound lysophosphatide acyltransferase. CSA, at concentrations identical with those inhibiting macromolecular synthesis, also inhibited the Con A-stimulated specific increase in the incorporation of labeled fatty acids into plasma membrane phospholipids. When lymphocytes were stimulated with Con A for 1 hr, incorporation of labeled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. CSA at a concentration of 200 ng/ml prevented the elevated incorporation of labeled fatty acids into plasma membrane phospholipids of Con A-stimulated thymocytes. Concomitantly, the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long-chain fatty acids into phospholipids, was strongly inhibited. Up to high concentrations, CSA had no effect on the phospholipid metabolism of unstimulated lymphocytes. The results suggest that CSA inhibits the activation of T lymphocytes by interfering with the early activation of plasma membrane phospholipid metabolism.     

Immunologic distinction of human muscle adenylate deaminase from the isozyme(s) in human peripheral blood cells: Implications for myoadenylate deaminase deficiency

Rabbit antisera to human muscle adenylate deaminase efficiently precipitate this enzyme from crude homogenates of muscle from man, monkey, and horse, without affecting other enzymes and proteins in the sample. They fail to react, however, with the adenylate deaminase of purified human erythrocytes, granulocytes, lymphocytes, and platelets. The exclusive presence of an antigenic determinant in the muscle adenylate deaminase suggests that this isozyme is determined by a unique gene. Since patients with myoadenylate deaminase deficiency have normal levels of the enzyme in their erythrocytes and leukocytes, this constitutes the strongest evidence at present that the deficiency state is due to an inherited gene defect.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

Changes in deoxyadenosine production during human lymphocyte mitogenesis

Immunodeficient children who lack the purine metabolic enzyme adenosine deaminase have markedly elevated plasma concentrations of 2'-deoxyadenosine and adenosine. However, little information exists concerning the magnitude of endogenous 2'-deoxyadenosine and adenosine synthesis by normal human hematopoietic cells. In the present experiments, we have used the sensitive technique of high performance liquid chromatography to quantitate changes in 2'-deoxyadenosine and adenosine production during human lymphocyte mitogenesis. In the presence of the adenosine deaminase inhibitor deoxycoformycin, human T lymphocytes stimulated with phytohemagglutinin (PHA), and non-T lymphocytes stimulated with formalinized Staphylococcus aureus Cowan strain I, excreted 2'-deoxyadenosine into the cell medium. The nucleoside was detectable as early as 20 h after addition of mitogen. The time course of 2'-deoxyadenosine excretion correlated with the uptake of [methyl-3H]thymidine into nucleic acid. Mitogen-stimulated human lymphocytes produced only minimal amounts of adenosine. The results suggest that increased 2'-deoxyadenosine synthesis and release may normally accompany human lymphocyte mitogenesis.     

Characterization of the adenosine deaminase-adenosine deaminase complexing protein binding reaction

Glutaraldehyde-fixed membranes from rabbit kidney cortex were used to characterize binding of monomeric adenosine deaminase to the adenosine deaminase complexing protein. With the use of bovine adenosine deaminase it was shown that enzyme binding is a saturable, high affinity process. The K value for binding of the bovine enzyme was 11 nM. Maximum enzyme binding and rate of binding to a constant amount of membrane did not vary significantly from pH 5.0 to 9.5. Metal ions, with the exception of Hg2+, sulfhydryl reagents, and other proteins had little or a slightly stimulatory effect on maximum binding. Mercuric ion inhibited binding. Using biotinylated bovine adenosine deaminase it was shown that purified rabbit, human, and monkey enzymes compete for binding sites on fixed membranes. The K values for the rabbit and human enzymes were 9 and 6 nM, respectively. Mouse or guinea pig adenosine deaminase did not bind to the membranes or compete with the biotinylated bovine enzyme for binding sites. The retention of characteristics required for binding by enzymes from rabbit, human, monkey, and calf tissues argues for biologic significance of the adenosine deaminase-complexing protein interaction. The basis for the apparent failure of rodent adenosine deaminase to bind to complexing protein remains to be determined.     

Adenosine deaminase activity in leukemia patients

The activities of adenosine deaminase (ADA) were measured in the blood plasma, erythrocytes, and lymphocytes of healthy reference persons and in patients affected with leukaemia. ADA is increased in patients with acute immature cell leukaemia, in patients with chronic lymphatic leukaemia it is comparatively low in lymphocytes. In chronic myeloic leukaemia ADA activities are different depending on the activity of the disease. ADA-activities in the blood plasma, erythrocytes, and lymphocytes do not correlate with each other. ADA-activities in leukaemias may be regarded as an indicator of increased purin metabolism rather a as parameter of disturbed cellular immunofunction.     

The potential importance of soluble deoxynucleotidase activity in mediating deoxyadenosine toxicity in human lymphoblasts

Deoxyadenosine and its nucleotides have been implicated in the pathogenesis of the immune dysfunction associated with a genetic deficiency of adenosine deaminase (ADA). We have previously shown that when ADA is blocked with a synthetic inhibitor, human T lymphoblastoid cell lines are more sensitive to deoxyadenosine toxicity, dephosphorylate deoxyadenosine nucleotides at a slower rate, and have much lower levels of ecto-5'-nucleotidase than most B cell lines. It seemed unlikely, however, that an enzyme on the outer surface of the lymphocyte plasma membrane could regulate intracellular deoxynucleotide catabolism. We now report that human lymphoblasts also contain a soluble deoxynucleotidase activity that is distinguishable from the plasma membrane enzyme by several criteria. In multiple human lymphoblastoid cell lines of varying origin and phenotype. soluble deoxynucleotidase correlated significantly (rs = 0.80, p < 0.001) with sensitivity to deoxyadenosine toxicity.     

Assessment of enzyme inhibition or stimulation in plasma membrane preparations

The purity of isolated plasma membranes is routinely judged by the activity of enzymes present both in this membrane and other locations in the cell. However, since enzyme inhibition and/or stimulation often occurs following disruption of the cell, the question as to which enzyme(s) provides a reliable marker of membrane purity should be considered. We have devised a simple method with which to address this problem. Inhibition or stimulation of plasma membrane marker enzymes can be rapidly assessed in cell homogenates and subfractions by mixing both samples, with known enzyme activity, and observing any deviation from the expected combined activity. Should the activity remain constant that enzyme can be used to gauge the purity of the plasma membrane preparation. Of the four putative plasma membrane marker enzymes examined only one, gamma-glutamyltranspeptidase appeared to give a reliable purity measurement in the cell system studied.     

ECTO-enzyme activity of human erythrocyte adenosine deaminase

Summary 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.Abbreviations ADA Adenosine Deaminase - LDH Lactate Dehydrogenase - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - CPR 6-Chloropurine Ribonucleoside - SDS Sodium Dodecyl Sulfate - NAD -Nicotinamide Adenine Dinucleotide - HBSS Hank's Balanced Salt Solution - DASA Diazotized Sulfanilic Acid     

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH

The isolated intact white adipocyte of the Swiss mouse responds to both ACTH and catecholamines by an elevation of cAMP levels and an increase in lipolysis. However, in the isolated plasma membrane of the mouse adipocyte, adenylate cyclase loses its responsiveness to ACTH but retains its ability to respond to catecholamines. This lack of responsiveness to ACTH by adenylate cyclase of mouse adipocyte plasma membrane can be overcome, at least partially, by addition of GPP (NH)p, an analog of GTP, to the assay medium. The data on mouse adipocyte membrane suggests that the coupling of ACTH receptor to adenylate cyclase is dependent on GTP and that catecholamine-activation of adenylate cyclase is less dependent on this nucleotide. The isolated intact white adipocyte of adult New Zealand rabbit responds to ACTH, but does not (or only weakly) respond to catecholamines. In contrast to the mouse plasma membrane preparation, adenylate cyclase of adipocyte membrane of the rabbit responds to ACTH. And the addition of GPP(NH)P is not required to demonstrate the CTH: sensitive adenylate cyclase activity. The difference between mouse and rabbit adipocyte membrane in the requirement for GPP(NH)P in ACTH action is not readily explained. The lack of catecholamine sensitivity of rabbit membrane enzyme cannot be reversed by addition of GPP(NH)P or adenosine deaminase. These two adenylate cyclase model systems using mouse and rabbit adipocyte plasma membrane may be useful tools for the study of the specificity and mechanism of action of lipolytic hormones such as ACTH and catecholamines.     

Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes

The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.     

Evaluation of methods for the isolation of plasma membranes displaying guanosine 5'-triphosphate-dependence for the regulation of adenylate cyclase activity: potential application to the study of other guanosine 5'-triphosphate-dependent transduction systems

The GTP-dependence for stimulatory and inhibitory regulation of plasma membrane adenylate cyclase activity was measured in plasma membrane fractions isolated from a variety of cell types (platelets, lymphocytes, PC12 cells, GH3 cells, NBP2 cells, and hepatocytes). This report shows that the isolation of plasma membranes for the study of GTP-dependent adenylate cyclase activity was, for some cells, enhanced by the exposure of the cells to glycerol prior to cell lysis. The isolation of plasma membranes from other cells, which did not appear to be sensitive to glycerol pretreatment, was enhanced by the removal of heavy particulate matter prior to fractionation of the cell lysate. The regulation of enzyme activity by various agents was found to be dependent upon the presence of (exogenous) GTP to varying degrees, indicating variable contamination of membrane preparations with GTP. It is concluded that (i) exposure of platelets and lymphocytes to glycerol prior to cell lysis decreases subsequent contamination of the plasma membrane preparation with GTP, and (ii) although glycerol pretreatment of other cells does not ensure the subsequent isolation of plasma membrane adenylate cyclase activity displaying high requirements for (exogenous) GTP, it is a reasonable first approach to be used during the development of procedures for the isolation of plasma membranes.     

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.     

Role of diacylglycerol kinase alpha in the attenuation of receptor signaling

Diacylglycerol kinase (DGK) is suggested to attenuate diacylglycerol-induced cell responses through the phosphorylation of this second messenger to phosphatidic acid. Here, we show that DGKalpha, an isoform highly expressed in T lymphocytes, translocates from cytosol to the plasma membrane in response to two different receptors known to elicit T cell activation responses: an ectopically expressed muscarinic type I receptor and the endogenous T cell receptor. Translocation in response to receptor stimulation is rapid, transient, and requires calcium and tyrosine kinase activation. DGKalpha-mediated phosphatidic acid generation allows dissociation of the enzyme from the plasma membrane and return to the cytosol, as demonstrated using a pharmacological inhibitor and a catalytically inactive version of the enzyme. The NH(2)-terminal domain of the protein is shown to be responsible for receptor-induced translocation and phosphatidic acid-mediated membrane dissociation. After examining induction of the T cell activation marker CD69 in cells expressing a constitutively active form of the enzyme, we present evidence of the negative regulation that DGKalpha exerts on diacylglycerol-derived cell responses. This study is the first to describe DGKalpha as an integral component of the signaling cascades that link plasma membrane receptors to nuclear responses.