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Two enzymes participating in 2'-deoxyadenosine (dAdo) metabolism: dAdo kinase (dAdoK EC 2.7.1.76) and adenosine deaminase (ADA, EC 3.5.4.4) were partially purified from rat liver mitochondria and cytosol and influence of nucleosides and nucleotides on the activity of these enzymes were investigated. Mitochondrial and cytosol dAdoK are separate proteins, while ADA from both subcellular fractions possesses similar physical properties. dGTP, a competitive inhibitor of mitochondrial dAdoK, inhibits cytosol ADA in a mixed way but activates mitochondrial ADA and cytosol dAdoK. A possible effect of dGTP on dAdo metabolism in mitochondria and cytosol is discussed.

Several adenosine analogs, such as coformycin, 2'-deoxycoformycin and erythro-9-(3-nonyl-p-aminobenzyl)adenine (EHNA), which are strong inhibitors of mammalian adenosine deaminase, are much weaker inhibitors of the Saccharomyces cerevisiae enzyme. The specificity of the yeast enzyme is more restricted than that of mammalian adenosine deaminase, particularly towards the ribose moiety and around position 6 and 1 of the substrate. The sulphydryl group appears to be more masked in the yeast than in the mammalian enzyme. The kinetic effects of pH with adenosine substrate and with the inhibitor purine riboside are reported. The findings on specificity and pH kinetic effects can be interpreted in a model involving proton transfer from the -SH group of the enzyme to the N-1 atom of the substrate.

The efficiency of retroviral-mediated gene transfer into hematopoietic stem cells (HSC) is dependent on the survival and self-renewal of HSC in vitro during retroviral infection. We have examined the effect of prestimulation of bone marrow with various cytokines, including the product of the Steel gene, Steel factor or stem cell factor (SCF) (the ligand for the c-kit receptor) on the efficiency of retroviral transduction of the human adenosine deaminase (hADA) cDNA into murine HSC. Bone marrow cells were prestimulated for 48 hours with hematopoietic growth factors, then cocultivated with the packaging cell line producing the ZipPGK-ADA simplified retrovirus for an additional 48 hours with continued growth factor exposure. Nonadherant cells from these cocultures were injected into lethally irradiated recipients. The content of day 12 colony-forming unit-spleen (CFU-S12) in SCF/interleukin 6 (IL-6)-prestimulated and cocultured bone marrow was more than threefold greater than that of IL-3/IL-6-prestimulated bone marrow cells. All mice receiving bone marrow cells infected with the PGK-ADA virus after prestimulation with IL-3/IL-6 or SCF/IL-6 demonstrated hADA expression in the peripheral blood after full hematopoietic reconstitution. While all recipients of IL-3/IL-6-prestimulated bone marrow expressed hADA at 4 months posttransplant, in three independent experiments examining a total of 33 mice, in most recipients of SCF/IL-6-prestimulated and infected bone marrow cells, the expression of human enzyme was higher than IL-3/IL-6 mice. Southern blot analysis of DNA from hematopoietic tissues from these same mice prepared at least 4 months posttransplantation also demonstrated a higher infection efficiency of HSC as measured by proviral integration patterns and genome copy number analysis. These results suggest that the higher level of hADA expression seen in mice receiving marrow prestimulated with SCF/IL-6 before retroviral infection is due to more efficient infection of reconstituting HSC. Other growth factor combinations were also studied; however, prestimulation with SCF/IL-6 or IL-3/IL-6 appeared optimal. Using retroviral-mediated gene transfer and viral integration patterns, Steel factor (SCF) in combination with IL-6 appears to increase the survival and self-renewal of reconstituting hematopoietic stem cells and proves useful in effecting expression of foreign genes in transplant recipients. Such pretreatment may also be useful in the application of retroviral transfer methods to human cells.

Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.

Adenosine deaminase (ADA) was partially purified 486- and 994-fold from rat liver mitochondria and cytosol, respectively. Relative molecular mass of the enzymes from both fractions was 34,000. Km for adenosine and 2'-deoxy-adenosine were 3.08 x 10(-5) M and 3.03 x 10(-5) M for mitochondrial ADA and 3.12 x 10(-5) M and 2.87 x 10(-5) M for cytosolic ADA. The enzyme from both subcellular fractions had the maximum activity at pH 7.5-8.0, and pI 5.2 and 4.2 for mitochondrial and cytosolic enzyme, respectively. The enzyme was inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine and 2'-deoxycoformycin with Ki 4.4 x 10(-7) M and 3.2 x 10(-7) M for mitochondrial ADA and 4.9 x 10(-7) M 2.8 x 10(-7) M for cytosolic ADA. Among the natural nucleoside and deoxynucleotide derivatives tested, deoxy-GTP and UTP inhibited only cytosolic adenosine deaminase by 60% and 40%, respectively.

Adenosine deaminase, which is essential for lymphoid differentiation and function, has previously been considered to be a cytosolic enzyme. In this report we demonstrate that it can be found associated with the plasma membrane of lymphocytes. By means of immunological techniques using both light and electron microscopy, adenosine deaminase was localized on the external side of the plasma membrane of normal lymphocytes and monocytes. Since the enzyme expression differed depending on the type of cell examined, new hypotheses about the mechanisms involving purine metabolism in immune dysfunctions or immunodeficiency syndromes may be considered.

Diethylaminoethyl-cellulose chromatography was used to separate the two isoenzymes of adenosine deaminase (EC3.5.4.4), adenosine deaminase1 (ADA1) and adenosine deaminase2 (ADA2), in human plasma. One hundred and fifteen purine base, nucleoside, and nucleotide analogs were tested as inhibitors of this partially purified preparation of ADA2. Coformycin and 2'-deoxycoformycin were by far the most potent inhibitors of this isoenzyme (apparent Ki values 20 and 19 nM, respectively). ADA2 was also inhibited by nebularine (apparent Ki 1.5 mM) but was resistant to the potent ADA1 inhibitor (+)-erythro-9(2-S-hydroxy-3-R-nonyl)adenine. alpha-D-Adenosine also inhibited ADA2, as did several halogenated purine and adenine base analogs. Structural requirements for the binding of purine analogs to ADA2 are presented which provide a general basis for the design of specific inhibitors of ADA2. Such inhibitors may be useful in studies designed to provide an understanding of the physiological role of ADA2 both in the normal state and in diseases such as human immunodeficiency virus-1 infection where levels in plasma are increased markedly.

1. The scallop enzyme was separated by DE52 ion-exchange chromatography into two forms with the same mol. wt of 38,000 and similar characteristics. 2. The enzyme was inactivated in the absence of dithiothreitol and complete reactivation was achieved by adding the agent within a critical storage period. 3. The apparent values of pKm and Vmax sensitively increased as ionic strength was raised to 250 mM and phosphate and sodium ions elevated the former value with a further increase of the ionic strength. 4. The apparent activation energies for the alpha (Vmax/Km) and beta (Vmax) parameters of both the forms were approximately 5 and 8 kcal/mol, respectively. 5. The enzyme deaminated 2'-, 3'-deoxyadenosine and 2',3'-isopropylidene adenosine but did not deaminate 5'-deoxyadenosine, alpha-adenosine and adenine nucleotides. 6. The affinity for inosine was much lowered with a high Ki value. Adenine and purine riboside inhibited the enzyme completely, and coformycin was a tight, slow binding inhibitor.

Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.

Lymphocyte populations of BALB/c mice were obtained from bone marrow, thymus, spleen, peripheral blood and lymphoid nodes. Subpopulations of thymocytes and bone marrow T-lymphocyte precursors were separated by density gradient centrifugation. The activity of adenosine deaminase (ADA) undergoes a marked increase during the evolution of bone marrow T-cell precursors to immature thymocytes, and a decrease with thymocytes maturation. The peripheral blood lymphocytes (PBL) present the lower activity of the enzyme, and lymphocytes from spleen (SL) and lymphoid nodes (LNL) show activity in the order of that in mature thymocytes. The activity of purine nucleotide phosphorylase (PNP) in the different lymphocytes populations experiments a very little variation with the T-lymphocyte differentiation. With the evolution of T-lymphocyte precursors to immature thymocytes the 5'-nucleotidase (5'-NT) activity experiment a 2-fold decrease. The thymocytes maturation is correlated with an increase in the activity of 5'-NT. The PBL present the maximal activity of the enzyme, whereas in spleen and LNL its levels of activity are in the range of that in mature thymocytes and bone marrow T-cell precursors respectively.

The small-size adenosine deaminase (Mr = 35,000 and 43,000) in calf intestinal mucosa, frog liver and scallop adductor muscle and the large-size deaminase (Mr = 100,000) in frog liver probably formed by adding a conversion protein to the small enzyme, were tightly bound to the purine riboside affinity column. Binding of the other large-size enzymes (Mr = 140,000) in the midgut gland of scallop and mussel to the column was not successful. It seems that the binding difference does not depend on a change in affinity between active site and ligand but rather on the stereospecific positioning of active site in the enzyme molecules.

Two fractions of adenosine deaminase (ADA) were separated by ion-exchange chromatography and purified to homogeneity from human thymus tissue by a combination of conventional biochemical methods and affinity chromatography. Some of the physical, chemical and serological properties of the two fractions were compared to those of erythrocyte ADA. All three proteins had apparent molecular weights of about 45,000. They exhibited similar amino acid composition, specific enzymatic activities, Km values for adenosine and antigenic activities as determined by radioimmunoassay. A small portion of ADA isolated from thymus did not bind to complexing protein whereas all of the erythrocyte ADA was bound by this protein. So far, this has been the only difference found between thymic and erythrocyte ADA.

Adenosine deaminase is a purine salvage enzyme that catalyzes the deamination of adenosine and deoxyadenosine. Deficiency of the enzyme activity is associated with T-cell and B-cell dysfunction. Mutant adenosine deaminase has been isolated from heterozygous and homozygous deficient lymphoblast cell lines with the aid of an affinity matrix consisting of coformycin (a potent inhibitor of the enzyme) as the affinity ligand, bound to 3,3'-iminobispropylamine-derivatized Sepharose. Routinely, 80-90% of adenosine deaminase in crude cell homogenates could be bound to the material. Adenosine deaminase was specifically eluted by enzyme inhibitors or less efficiently by high substrate concentrations. Protein preparations isolated from several different deficient cell lines were highly purified and exhibited molecular weights identical to wild-type adenosine deaminase. This method produces a protein that is suitable for structural studies.

Red cell adenosine deaminase from normal subjects and from a patient with hereditary hemolytic anemia with a 40-fold increase in activity were purified using antibody affinity chromatography. The purified enzymes were completely homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were no differences in the molecular weight, specific activity, polyacrylamide gel electrophoresis, Michaelis constant for adenosine, inhibition constant for guanylurea, utilization of 2-deoxyadenosine, thermal stability, optimum pH, immunological reactivity, amino acid composition and tryptic peptide mapping. These results strongly suggest that increased red cell adenosine deaminase activity is caused by an overproduction of a structurally normal enzyme.

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.

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.

Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified in high yield from human erythrocytes using a simple procedure based on two chromatographic steps. In the first step enzyme is bound to a diaminohexane-Sepharose column and eluted with salt. The second strap is column chromatography on Separose-bound purine riboside prepared by cross-linking the riboside to the matrix with 1,4-butanediol-diglycidyl ether. The overall purification factor was approximately 750,000 and the purity was at least 96%. The procedure is suitable for small sample sizes and for purification of the variant forms of the enzyme obtained from a single individual.

1. The adenosine deaminase has an approximate molecular weight of 130,000-140,000 and the composition of two polypeptide units (mol. wt about 68,000) is suggested, by means of SDS disc electrophoresis. 2. Both the alpha (Vm/Km) and beta (Vm) parameters were varied with pH and temperature. RSS (relative substrate specificity) adenosine and deoxyadenosine values for alpha and beta were 1.2 and 1.1, respectively. 3. Adenine, 2'-, 3', 5'-AMP, 5'-deoxyAMP, ADP and ATP were not deaminated by the enzyme. 4. Inhibition by Mg2+ was found in reaction with adenosine at pH 8 but not with deoxyadenosine at the same pH. Mn2+, which did not affect the reaction rate at pH 4 and 5, showed competitive inhibitory effects at pH 6, 7 and 8.

Adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) has been purified 468,000-fold from pooled human erythrocytes. The procedure developed was used to isolate the enzyme from up to 23 liters of packed erythrocytes at one time. An easily prepared affinity column bed material employing adenosine as the ligand was used as the final step in the purification. During elution from the affinity column there was approximately a 3:1 partition of adenosine deaminase between gel bed and column buffer. There was no apparent difference in the partitioning of unresolved or partially resolved preparations of the electrophoretically different forms of the enzyme on the affinity column. Gel filtration and electrophoresis on polyacrylamide gels of increasing concentration revealed no differences in the Mr of these electrophoretically different forms. The four bands resolved by electrophoresis of the different forms on polyacrylamide gels under nondenaturing conditions yielded a single band when electrophoresis was carried out in the presence of sodium dodecyl sulfate and 2-mercaptoethanol. Partially resolved preparations of the different electrophoretic forms of adenosine deaminase also gave rise to a single band of the same mobility when electrophoresed on polyacrylamide gels under these conditions. The band had the mobility of a protein of Mr of 36,000. This Mr is approximately the same as estimated for the nondenatured enzyme.

Adenosine deaminase (ADA) was partially purified from several sources using affinity chromatography. These enzymes have the capacity to catalyze the deamination of 2,6-diamino-9-(2-hydroxyethoxymethyl)purine (A134U) to form the antiviral agent acyclovir [9-(2-hydroxyethoxymethyl)guanine]. Their relative substrate efficiencies (Vmax/Km) with A134U (standardized to adenosine = 100) were: dog ADA, 0.092; human ADA, 0.015-0.029; rat ADA, 0.025; calf ADA, 0.016; and Escherichia coli ADA, 0.0003. In addition to having the lowest efficiency with A134U, the bacterial ADA was also distinguished by its lack of binding of the mammalian ADA inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine and by its weak binding to the 9-(p-aminobenzyl)adenine-agarose affinity column. Four minor metabolites of A134U and acyclovir have been reported to be produced in the rat. These compounds are oxidized on either the C-8 position of the ring or the terminal carbon of the side chain. Neither acyclovir nor any of these metabolites produced significant inhibition of calf intestine ADA. The oxidized metabolites containing an N-6 amino group were extremely slow substrates of this enzyme.

1. The A and C forms of bovine liver adenosine deaminase (adenosine aminohydrolase; EC 3.5,4.4) have been separated. 2. The proportion of two forms is dependent on ionic strength of solution. 3. By gel filtration, in presence of 6 M urea, and A form is dissociated into the C form and the binding factor and both are also separated. By removal of urea the A form is again obtained. 4. The molecular weights of two forms and binding factor, kinetic parameters have been determined.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol. The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations. Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature. B. cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM.

Several reports have suggested that a decrease or absence of adenosine deaminase complexing protein (ADCP) is consistently associated with cancer. However, in other studies, decreased as well as increased ADCP levels were found. In the present study, we investigated ADCP levels in 37 colorectal adenocarcinomas and correlated the results with clinicopathological characteristics in individual carcinomas. The levels of adenosine deaminase (EC 3.5.4.4) and soluble ADCP were determined in tissue samples by, respectively, a spectrophotometric assay and an ADCP specific radioimmunoassay. The values in the individual tumors were compared with their histological characteristics, such as degree of differentiation, nuclear grading, and the preoperative plasma carcinoembryonic antigen levels in the patients. It was found that ADCP was decreased in about a third of the tumors but unaltered or even increased in others. However, there was an overall 40% increase of the adenosine deaminase activity in the tumors compared to normal tissue. There seems to be no simple correlation between any of the clinicopathological parameters and the ADCP or adenosine deaminase levels. Methods detecting ADCP at single cell level might be helpful in exploring its potential use as a cancer-associated marker.

The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

A protein which specifically complexes with adenosine deaminase (complexing protein) has been purified to homogeneity from human plasma. This protein was compared with complexing protein isolated from human kidney. The two proteins produce electrophoretically different forms of high molecular weight adenosine deaminase when combined with the Mr = 36,000 enzyme monomer from erythrocytes. This difference may, at least in part, be due to the greater sialic acid content of complexing protein from plasma. By other criteria, including amino acid composition, total carbohydrate content, and subunit structure, the two proteins are quite similar. In addition, plasma complexing protein shows complete cross-reactivity with anti-kidney complexing protein serum. These results suggest that plasma and kidney complexing proteins are products of the same gene.

Adenosine deaminase (EC 3.5.4.4) was found to occur in the extract of Azotobacter vinelandii, strain 0, and purified by heating at 65 degrees C, fractionation with ammonium sulfate, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. Purified adenosine deaminase was effectively stabilized by the addition of ethylene glycol. The molecular weight of the enzyme was estimated to be 66,000 by gel filtration on Sephadex G-150. The enzyme specifically attacked adenosine and 2'-deoxyadenosine to the same extent, and formycin A to a lesser extent. The pH optimum of the enzyme was observed at pH 7.2. Double reciprocal plot of initial velocity versus adenosine concentration was concave upward, and Hill interaction coefficient was calculated to be 1.5, suggesting the allosteric binding of the substrate. ATP inhibited adenosine deaminase in an allosteric manner, whereas other nucleotides were without effect. The physiological significance of the enzyme was discussed in relation to salvage pathway of purine nucleotides.

Rat brain adenosine deaminase (E.C. 3.5.4.4.) was purified 667-fold from the supernatant fraction by the following techniques: heat treatment (60 degrees C), fractionation with ammonium sulfate, column chromatography on DEAE-Sepharose, and preparative gel electrophoresis. The purified enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and isoelectric focusing. Amino acid composition is given. The isoelectric point of the enzyme (5.2) was determined by isoelectric focusing on agarose. The apparent molecular weight was estimated to be 39,000 (Stokes Radius [Rs] = 27.3 A) using a calibrated Sephacryl S-300 column. The study of the influence of the temperature on the initial reaction rates allowed calculation of Ea (8.9 Kcal/mole) and delta H (5.0 Kcal/mole) values. The variation of V and Km with pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme-substrate complex. The enzyme had a Km (adenosine) of 4.5 X 10(-5) M and was inhibited by inosine, guanosine, adenine, and hypoxanthine but not by other intermediates of purine metabolism. None of the inhibitors were active as substrates. The enzyme was also inhibited by dimethyl sulfoxide and ethanol. Inhibition by ethanol can account partially for the CNS depressant effects of levels 3 and 4 of alcohol intoxication. A number of drugs having therapeutic uses such as sedative, anxiolytic, analgesic, and relaxant are modulators of the enzyme. Among these, lidoflazine, phenylbutazone, and chlordiazepoxide are the most potent as inhibitors (Ki 30, 54, and 83 microM, respectively), whereas medazepam is the most potent as activator (Ka 0.32 mM). Thus, it is concluded that some drugs that inhibit adenosine uptake also modulate adenosine deaminase activity. Besides, since the enzyme is located extracellularly [Franco et al, 1986], these drugs can modulate the physiological effects exerted by extracellular adenosine.

Immobilised inosine (6a) and adenosine (6c) and their 5'-phosphates have been synthesized. Reaction of the nucleosides with ethyl levulinate, followed by saponification or phosphorylation and then saponification, gave the 2',3'-O-[1-(2-carboxyethyl)ethylidene] derivatives 3 and 4 and the corresponding 5'-phosphates 2b and 2d. 6-Aminohexylagarose (5) was severally coupled to 2b, 2d, 3, and 4 through the carboxyl groups to give the polymers 6a-d. Adenosine deaminase converts 3 into 4, and 6c into 6a. The polymers can be used as affinity resins for adenosine deaminase, which is bound more strongly to 6c than to 6a. The operational capacity of 6a for adenosine deaminase is constant at 15--25 degrees, but decreases by approximately 16% from 25 degrees to 35 degrees. The resin 6a has been used to separate adenosine deaminase from mixtures containing other enzymes, for example, guanase or alcohol dehydrogenase.

Adenosine aminohydrolase from monkey brain was purified ten fold. In pH 7.3 phosphate buffer at 37 degrees, this enzyme preparation deaminated adenosine and arabinosyladenine with apparent values for the Michaelis constant of 32 muM and 370 muM respectively. The products of both deamination reactions, i.e., inosine and arabinosylhypoxanthine, were competitive inhibitors with Ki equal to 220 muM and 1,000 muM, respectively. N6-benzyladenosine and 9-(1-hydroxy-2-octyl)adenine were competitive inhibitors and were more effective in inhibiting deamination of arabinosyladenine than of adenosine. It is suggested that these compounds might potentiate arabinosyladenine chemotherapy of neoplasms of the central nervous system.

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.

A low molecular weight form of adenosine deaminase from bovine skeletal muscle was purified about 930-fold. The enzyme had a mol. wt of 31,000, a Km value for adenosine of 2.37 X 10(-5) M and a pH optimum at 7.0. This enzyme is very resistant to heat inactivation and does not require metal activators or other dialysable cofactors. A possible role in the post-mortem metabolism of adenine nucleotide in skeletal muscle is discussed.

A deficiency of the enzyme adenosine deaminase is associated with an autosomal recessive form of severe combined immunodeficiency disease in man. The molecular forms of the normal human enzyme have now been well characterized in an effort to better understand the nature of the enzyme defect in affected patients. In some human tissues adenosine deaminase exists predominantly as a small molecular form while in other tissues a large form composed of adenosine deaminase (small form) and an adenosine deaminase-binding protein predominates. The small form of the enzyme purified to homogeneity by antibody affinity chromatography is a monomer of native molecular weight of 37,600. The adenosine deaminase-binding protein, purified by adenosine deaminase affinity chromatography, appears to be a dimer of native molecular weight 213,000 and contains carbohydrate. Based on direct binding measurements, chemical cross-linking studies and sedimentation equilibrium analyses, small form adenosine deaminase has been shown to combine with purified binding protein in a molar ratio of 2:1 respectively to produce the large form adenosine deaminase. Reduced, but widely ranging levels of adenosine deaminating activity, have been reported in various tissues of adenosine deaminase deficient patients. Further, the characteristics of this residual enzyme activity have been analyzed immunochemically to substantiate genetic heterogeneity in this disorder. While many types of immunodeficiency are currently recognized in man, in most cases the molecular defect is unknown. The discovery of a deficiency of the enzyme, adenosine deaminase, ADA, (EC 3.5.4.4), in some patients with severe combined immunodeficiency disease represented an early clue to the pathogenesis of immune dysfunction at the molecular level 1-4. Affected patients with markedly reduced levels of ADA exhibit a defect of both cellular and humoral immunity characterized clinically by severe recurrent infections with a fatal outcome if untreated. Attempts to elucidate the nature of the genetic mutation(s) leading to the reduction of ADA activity in these immunodeficient patients have been complicated in part by an incomplete understanding of the nature of ADA in normal tissues. In this review we will consider the structural characteristics of the normal and mutant forms of ADA as they are currently understood.

Chicken egg yolk contains an adenosine deaminase that was investigated after purifying about 500 times. It has a pH optimum at 6.5, aKm of 6.6 times 10(-5) mol/l and an approximate molecular weight of 14000; higher molecular forms could not be detected. It was compared with the adenosine deaminases of chicken liver and blood plasma. From this comparison it is evident that this protein has undergone certain changes during the successive events leading to its final structure (secretion by the liver, transport through blood plasma to the oocytes and development of the egg): a common subunit with an approximate molecular weight of 15000 may be the basis of the physiological diversifications. Substrate specificity of the purified extracts extends to cytidine and guanosine also, although certain observations point to different enzymes being involved. Deoxyadenosine is also deaminated. Cu2+, Zn2+, and Pb2+ are inhibiting and free -SH seems essential for activity.

Human malaria infected erythrocytes show a dramatic increase in adenosine deaminase activity in vitro. Using recently developed culture techniques, adenosine deaminase-deficient human erythrocytes were infected in vitro with the major human pathogen Plasmodium falciparum. Adenosine deaminase activity was undetectable in the uninfected host red cells, but increased by 2-fold over normal levels in these cells with an 8% parasitemia. The enzyme in these cells appeared unique in that its activity was markedly elevated over that of other parasite purine enzymes, was not cross-reactive with antibody against human erythrocyte adenosine deaminase, and though inhibited competitively by deoxycoformycin was relatively insensitive to erythro-9-(2-hydroxy-3-nonyl) adenine. The use of adenosine deaminase-deficient erythrocytes for the in vitro cultivation of Plasmodium provides a unique system for the study of parasite enzyme and allows further insight into the purine metabolism of the intraerythrocytic malaria parasite.

Two forms of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), differing in molecular size, have been purified and obtained in homogeneous form from rabbit intestine. The purification procedures involved extraction with acetate buffer, pH 5.5, precipitation and fractional reextraction with (NH4)2SO4, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 and Sephadex G-200. Gel filtrations analysis gave molecular weight estimates of 265 000 and 32 000 for the large and small deaminases respectively. The two enzymes forms had similar pH optima and pH stability ranges.