Amplification and molecular cloning of murine adenosine deaminase gene sequences

A previously isolated mouse Cl-1D derived cell line (B-1/25) overproduces adenosine deaminase (EC 3.5.4.4) by 3200-fold. The present studies were undertaken to determine the molecular basis of this phenomenon. Rabbit reticulocyte lysate and Xenopus oocyte translation studies indicated that the B-1/25 cells also overproduced adenosine deaminase mRNA. Total poly(A+) RNA derived from B-1/25 was used to construct a cDNA library. After prehybridization with excess parental Cl-1D RNA to selectively prehybridize nonamplified sequences, 32P-labeled cDNA probe synthesized from B-1/25 total poly(A+) RNA was used to identify recombinant colonies containing amplified mRNA sequences. Positive clones containing adenosine deaminase gene sequences were identified by blot hybridization analysis and hybridization-selected translation in both rabbit reticulocyte lysate and Xenopus oocyte translation systems. Adenosine deaminase cDNA clones hybridized with three poly(A+) RNA species of 1.5, 1.7, and 5.2 kilobases in length, all of which were overproduced in the B-1/25 cell line. Dot blot hybridization analysis using an adenosine deaminase cDNA clone showed that the elevated adenosine deaminase level in the B-1/25 cells was fully accounted for by an increase in adenosine deaminase gene copy number. The adenosine deaminase cDNA probes and the cell lines with amplified adenosine deaminase genes should prove extremely useful in studying the structure and regulation of the adenosine deaminase gene.     

Increased adenosine deaminase synthesis and messenger RNA activity in deoxycoformycin-resistant cells

The basis for the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells was investigated. Three variant cell lines with different levels of adenosine deaminase activity showed increases in the relative rate of synthesis of the enzyme in vivo. No difference in the rate of degradation of the enzyme was seen between the parental cell line and one variant cell line which exhibits a 180-fold increase in adenosine deaminase activity. Polysomal RNA isolated from this variant exhibited a 175-fold increase in the ability to direct the synthesis of adenosine deaminase in vitro.     

Adenosine deaminase from deoxycoformycin-sensitive and -resistant rat hepatoma cells. Purification and characterization

Deoxycoformycin-resistant rat hepatoma cells exhibit up to 300-fold increase in adenosine deaminase activity compared to the sensitive parental cells. In order to determine the basis of the increased enzyme activity in deoxycoformycin-resistant cells, adenosine deaminase was purified from rat liver and deoxycoformycin-sensitive and -resistant cells. Physical, kinetic, and immunological properties of the purified enzymes were compared. Purified adenosine deaminase from all sources was found to be a monomer with an Mr approximately 45,000. In addition, the purified enzymes had a similar isozyme pattern in nondenaturing polyacrylamide gels. Km values for adenosine and Ki values for deoxycoformycin did not differ among the purified enzymes. By double diffusion analysis and quantitative immunoprecipitation, the purified enzymes were found to be immunologically indistinguishable. These data indicate that deoxycoformycin-resistant rat hepatoma cells produce increased amounts of adenosine deaminase protein which results in increased enzymatic activity.     

Amplification of adenosine deaminase gene sequences in deoxycoformycin-resistant rat hepatoma cells

Deoxycoformycin-resistant rat hepatoma cells exhibit up to a 2000-fold increase in adenosine deaminase activity compared to the sensitive parental cells. The increased enzyme activity in these cells is accompanied by similar increases in 1) the amount of adenosine deaminase protein, 2) the relative rate of adenosine deaminase synthesis in vivo, and 3) adenosine deaminase mRNA activity. To further investigate the mechanism(s) responsible for the overproduction of adenosine deaminase in these cells, we have isolated a recombinant plasmid containing a 1.4-kilobase insert complementary to at least part of the adenosine deaminase mRNA. Using this cDNA as a specific hybridization probe, all deoxycoformycin-resistant variants were shown to have increased amounts of adenosine deaminase mRNA and gene sequences. The relative increase in the level of mRNA and gene copy number was similar to the relative increase in enzyme activity for most resistant cell lines. However, the degree of adenosine deaminase gene amplification in one deoxycoformycin-resistant cell line (6-10-200) was 3-4-fold less than the relative increase in adenosine deaminase mRNA. These results indicate that the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells is due in large part, but not exclusively, to gene amplification.     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

Genetic expression in partial adenosine deaminase deficiency. mRNA levels and protein turnover for the enzyme variants in human B-lymphoblast cell lines

A severe genetic deficiency of adenosine deaminase is causally associated with an autosomal recessive form of severe combined immunodeficiency disease, while subjects with absent erythrocyte but partial lymphocyte enzyme activity remain immunocompetent. The genetic expression of adenosine deaminase in B-lymphoblast cell lines derived from four unrelated subjects with the "partial" enzyme deficiency was examined. Enzymatic activity among these cell lines ranged from 5 to 50% of normal with the level of immunoreactive adenosine deaminase protein either proportional to enzyme activity or elevated in two of the cases. Northern blot analysis using a cDNA probe showed that adenosine deaminase mRNA in each of these cell lines was of normal expected size (1.6-1.8 kilobases) and was present in normal to above normal amounts. Rates of enzyme synthesis varied from 165 to 15% of normal. Adenosine deaminase protein degradation rates in these cell lines were 1.5 to almost 3 times faster than normal, consistent with the observed absence of the enzyme in erythrocytes. From these analyses apparent abnormalities in mRNA regulation, translation, and protein degradation can be identified among the partially adenosine deaminase-deficient cell lines studied. Ultimately, it will be essential to determine the nature of the protein mutation and the gene defect to define the structural alterations and functional abnormalities of enzyme variants isolated from subjects with partial adenosine deaminase deficiency.     

Increased expression of one of two adenosine deaminase alleles in a human choriocarcinoma cell line following selection with adenine nucleosides

JEG-3 is a human choriocarcinoma cell line characterized by low levels of adenosine deaminase expression. For the purpose of studying adenosine deaminase gene regulation in the JEG-3 cells, we attempted to select variant cells having increased adenosine deaminase expression. This was accomplished by selecting cells for resistance to the cytotoxic adenosine analogs 9-beta-D-arabinofuranosyl adenine (ara-A) or 9-beta-D-xylofuranosyl adenine (xyl-A), both of which could presumably be detoxified by the action of adenosine deaminase. Single step high dose selection was ineffective in obtaining cells with increased adenosine deaminase. However, multistep selection using either ara-A or xyl-A resulted in cell populations with increased adenosine deaminase activity. Removal of selective pressure resulted in decreased adenosine deaminase levels. Subclones of xyl-A-resistant cells belonged to one of three phenotypic classes characterized by either elevated adenosine deaminase levels, decreased adenosine kinase levels, or both of these features. One subclone (A3-1A7) with unaltered adenosine kinase expression showed a 20-fold increase in adenosine deaminase expression. Further selection of this subclone for increasing xyl-A resistance resulted in an additional 2-fold increase in adenosine deaminase expression, followed by loss of adenosine kinase expression. These adenosine kinase-deficient cells showed no subsequent increase in adenosine deaminase expression in response to further xyl-A selection pressure. These results confirmed that xyl-A toxicity was mediated through its phosphorylated form and indicated that resistance may result from increased adenosine deaminase levels and/or adenosine kinase deficiency. The increased adenosine deaminase expression of the A3-1A7 subclone was exclusively in the ADA 2 allelic form. However, cell fusion experiments between A3-1A7 cells and mouse C1-1D cells established the existence of functional copies of both ADA 1 and ADA 2 allelic genes in the A3-1A7 cells. The increased expression of only one of the two functional ADA alleles, the requirement for a stepwise selection protocol to obtain cells with increased adenosine deaminase, and the instability of the adenosine deaminase phenotype in the absence of selective pressure suggest that the alteration of adenosine deaminase phenotype in the drug-resistant cells was the result of adenosine deaminase gene amplification.     

Purification of Adenosine Deaminase from Chicken-Egg Yolk by Affinity Column Chromatography

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.     

Purification of adenosine deaminase from chicken-egg yolk by affinity column chromatography

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.     

Complete release of adenosine deaminase from mouse lymphocytes stabilized by low-pH acetate

Complete release of adenosine deaminase from mouse lymphocytes takes place when intact cells are stabilized by low-pH acetate buffer. Both the low pH and the acetate affect the enzyme extraction markedly. At pH 5.0 all the adenosine deaminase activity detectable in the whole cell homogenates is released into the acetate buffer in very few minutes, with a total amount of 2% protein being extracted. The complete extraction of the enzyme activity is never observed when, at pH 5.0, the acetate is replaced by glutamate, citrate, succinate or maleate and only 45% and 15% of the adenosine deaminase activity is extracted by the acetate at pH 6.0 and 7.0, respectively. The breakdown of adenosine by the enzyme activity extracted from the stabilized cells is due to deamination alone, since inosine is the only product of the catalyzed reaction and its formation is completely inhibited by coformycin, a selective inhibitor of adenosine deaminase. The enzyme extracted shows a specific activity 50-times higher than that found in the crude homogenates, and a substantial purification of the enzyme extracted is achieved by a single Sephadex G-100 gel filtration.     

Purification and characterization of adenosine deaminase from Klebsiella sp. LF 1202

Adenosine deaminase was induced when the cells of Klebsiella sp. LF 1202 were cultured in the medium containing adenosine as a sole source of carbon and nitrogen. The induction was partially repressed by the addition of ammonium sulfate in the medium. The amount of adenosine deaminase reached approximately 4.6% of the total intracellular soluble proteins. The enzyme was purified approximately 22-fold with a 25% activity yield. The enzyme was a monomer with a molecular weight of 26,000. The optimal activity was obtained at pH 8.0, 37°C, and the K_m value for adenosine was 37 μM. Metal ions such as Zn²⁺, Co²⁺, Fe² and Ni⁺ inhibited the activity of the enzyme. Sulfhydryl blocking agents such as p-chloromercuribenzoate and HgCl₂ were also found to be potent inhibitors for adenosine deaminase.     

Isolation of mutant adenosine deaminase by coformycin affinity chromatography

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.     

Partial purification and characterization of S-adenosylhomocysteine hydrolase isolated from Saccharomyces cerevisiae

S-Adenosylhomocysteine (SAH) hydrolase was purified 25-fold from bakers' yeast by chemical methods and column chromatography. The purified enzyme could readily synthesize SAH from adenosine and homocysteine, but could hydrolyze only negligible amounts of SAH. The purified enzyme showed no activity towards S-adenosylmethionine, methylthioadenosine, or adenosine. Several nucleotides, sulfhydryl compounds, and ribose could not replace adenosine or homocysteine in the reaction mixture. SAH could be hydrolyzed by SAH hydrolase if commercial adenosine deaminase was included in the reaction mixture. Under these conditions l-homocysteine could act as a product inhibitor. A number of compounds structurally similar to adenosine and homocysteine were found to inhibit synthesis of SAH from adenosine and homocysteine. The strongest inhibitors were adenine, adenosine-3'-monophosphate, adenosine-2'-monophosphate, adenosine diphosphate, adenosine triphosphate, and adenosine-5'-monophosphate. The biosynthetic and hydrolytic activity of SAH hydrolase in yeast cell ghosts was similar to the activity of the enzyme in vitro.     

Human adenosine deaminase. Purification and subunit structure.

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

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.     

The measurement of membrane potential during photosynthesis and during respiration in intact cells of Rhodopseudomonas capsulata by both electrochromism and by permeant ion redistribution.

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.     

Purification and characterization of adenosine deaminase from camel skeletal muscle

Adenosine deaminase was purified (780-fold) from skeletal muscle of camel (Camelus Dormedarius) to homogeneity level by using DEAE Sephadex chromatography, ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The enzyme appeared to be monomeric with subunit molecular weight of 43kDa and isoelectric point of 4.85. The enzyme showed specificity for adenosine and exhibited Michaelis-Menten Kinetics with kappa(cat) of 1112.41 min(-1) and K(m) of 14.7 microM at pH 7.5. The pH and temperature optima for enzyme activity were 7-7.5 and 25 degrees C, respectively. Free energy (DeltaG*), enthalpy (DeltaH*) and entropy (DeltaS*) of activation for denaturation of adenosine deaminase at 50 degrees C were 88.94, 99.65 kJmol(-1) and 33.16 Jmol(-1), respectively. The purified enzyme had half-lives of 636 and 61 min at 25 and 50 degrees C, respectively. The activation energy for catalysis of camel skeletal muscle adenosine deaminase was 9.13 kJmol(-1). Free energy (DeltaG#), enthalpy (DeltaH#) and entropy (DeltaS#) of activation for hydrolysis of adenosine deaminase at 25 degrees C were 50.35, 6.65 kJmol(-1) and -146.62 Jmol(-1), respectively. Purine riboside inhibited the enzyme competitively with K(i) of 16 microM.     

Bromodeoxyuridine induction of deoxycytidine deaminase activity in a hamster cell line

The Syrian hamster cell line, RPMI 3460, was found to express barely detectable levels of the enzyme deoxycytidine deaminase. In contrast, the cell lines B4 and HAB, which are derived from 3460 cells and have approx. 60 and 100% bromodeoxyuridine substitution in DNA, respectively, show an approx. 50-fold higher enzyme activity. Deoxycytidine deaminase activity can be "induced" in 3460 cells by growth in 10(-5) M bromodeoxyuridine, as well as by the other halogenated pyrimidines, iododeoxyuridine and chlorodeoxy-uridine. The time required for maximal enzyme activity to accrue (approx. 8 days) suggests that new genetic expression is required for enhanced deoxycytidine deaminase activity and inhibition of induction in the presence of Ara. C shows that bromodeoxyuridine must be incorporated into DNA. In addition, the extent of enhanced deoxycytidine deaminase activity is directly related to the level of bromodeoxyuridine substitution in DNA. Another hamster cell line, BHK21/C13, which shows no detectable deoxycytidine deaminase activity, cannot be induced by bromodeoxyuridine. These results are discussed with respect to a mechanism by which bromodeoxyuridine may alter gene expression due to an altered binding of both positive and negative regulatory proteins to DNA.     

The antigen identified by a mouse monoclonal antibody raised against human renal cancer cells is the adenosine deaminase binding protein

The antigen recognized by a mouse monoclonal antibody (mAb S27) raised against a human renal cancer cell line has been identified as the adenosine deaminase binding protein. mAb S27 immunoprecipitates binding protein purified from a soluble fraction of human kidney. It also recognizes the mature 120,000-dalton membrane form of binding protein from [35S]methionine-labeled human fibroblasts, HepG2 cells, and the renal cancer cell line against which the antibody was raised. A rabbit polyclonal antibody raised against purified kidney binding protein completely precipitates mAb S27-reactive material from labeled membrane extracts. mAb S27 does not precipitate the initially synthesized 110,000 molecular weight precursor of binding protein in fibroblasts and recognizes only a small portion of binding protein precursor in labeled HepG2 cells suggesting that the antigenic determinant recognized by mAb S27 may be a post-translational modification present on the mature form of binding protein or that mAb S27 recognizes molecules in a certain conformation. Glycopeptides derived from purified soluble kidney binding protein or exogenously added adenosine deaminase do not inhibit the immunoprecipitation of binding protein by mAb S27, indicating that the mature oligosaccharide chains of binding protein are not the determinant recognized by mAb S27 and that bound adenosine deaminase does not mask the antigenic sites on binding protein. The fact that monoclonal antibody S27, previously shown (Ueda, R., Ogata, S., Morissey, D. M., Finstad, C. L., Szkudlavek, J., Whitmore, W. F., Oettgen, H. F., Lloyd, K. O., and Old, L. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5122-5126) to detect a cell surface antigen on cultured renal cancer cells, is directed against the adenosine deaminase binding protein confirms and extends the earlier observation (Andy, R.J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925) that binding protein is located on the cell surface.