Adenosine kinase from human liver.

Adenosine kinase (ATP: adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to homogeneity from human liver. The yield was 55% of the initial activity with a final specific activity of 6.3 mumol/min per mg protein. The molecular weight was estimated as about 40 000 by Sephadex G-100 gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 18% of that of adenosine. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad optimum at pH 7.5--8.5. The Km value for adenosine was 0.15 micrometer, and the activity was strongly inhibited at higher concentrations than 0.5 micrometer. ATP, dATP, GTP and dGTP were proved to be effective phosphate donors. Co2+ was more effective than Mg2+, and Ca2+, Mn2+, Fe2+ and Ni2+ showed about 50% of the activity for Mg2+. Some difference in structure between the adenosine kinase from human liver and that from rabbit or rat tissue, was observed by amino acid analysis and peptide mapping analysis.

Purification and properties of adenosine kinase from rat brain.

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to apparent homogeneity from rat brain by (NH4)2SO4 fractionation, affinity chromatography on AMP-Sepharose 4B, gel filtration with Sephadex G-100, and DE-52 cellulose column chromatography. The yield was 56% of the initial activity with a final specific activity of 7.8 mumol/min per mg protein. The molecular weight was estimated as 38 000 by gel filtration with Sephadex G-100 and 41 000 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 20% that of adenosine phosphorylation. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad pH optimum at pH 7.5-8.5. The Km value for adenosine was 0.2 microM and the maximum activity was observed at 0.5 microM. At higher concentrations of adenosine, the activity was strongly inhibited. The Km value for ATP was 0.02 mM and that for Mg2+ was 0.1 mM. GTP, dGTP, dATP and UTP were also proved to be effective phosphate donors. Co2+ was as effective as Mg2+, and Ca2+, Mn2+ or Ni2+ showed about 50% of the activity for Mg2+. The kinase is quite unstable, but stable in the presence of a high concentration of salt; e.g., 0.15 M KCl.

Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis.

Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 A using synchrotron radiation. The crystal belonged to space group P3(1)21, with unit-cell parameters a = 70.2, c = 111.6 A, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.

Cytokinin affinity purification and identification of a tobacco BY-2 adenosine kinase.

Adenosine kinase is one of the enzymes potentially responsible for the formation of cytokinin nucleotides in plants. Using a zeatin affinity column a 40 kDa protein was isolated from tobacco Bright Yellow 2 (TBY-2) and identified by mass spectrometry as adenosine kinase. The ligand interaction reported here can be disrupted by several other adenine- but not guanine-based purine derivatives. The observed interaction with cytokinins is discussed in view of a putative role for adenosine kinase in TBY-2 cytokinin metabolism. The presented results show for the first time a plant adenosine kinase affinity-purified to homogeneity that was identified by primary structure analysis.

Purification, characterization, substrate and inhibitor specificity of adenosine kinase from several Eimeria species.

Ribonucleosides of some pyrazolo [3,4-d] pyrimidines have been shown to be potent anticoccidial agents. To investigate their interactions with adenosine kinase, this enzyme was purified by affinity chromatography from the sporulated oocysts of 3 avian coccidia, Eimeria tenella, E. acervulina and E. brunetti as well as from chicken liver. Comparative studies revealed several differences among the enzymes. Magnesium appeared not to be inhibitor of the E. tenella enzyme but did inhibit the enzymes from the other three sources. ATP in excess of the magnesium concentration strongly inhibited the E. brunetti enzyme but had only a small effect on the other enzymes. The chicken liver enzyme utilized a broader variety of triphosphate donors than did any of the enzymes from Eimeria species. ATP, dATP, GTP, dGTP and ITP was the best substrates. Studies with pyrazolo [3,4-d] pyrimidine nucleosides revealed two groups of enzymes with similar inhibitor specificities, the chicken liver and E. Acervulina vs. the E. tenella and E. brunetti enzyme. This grouping roughly correlates with the in vivo anticoccidial specificity of these compounds. Substrate specificity studies using two 4-substituted pyrazolo [3,4-d] pyrimidine ribonucleosides (ethylthio- and cinnamylthio-), which have shown potent anticoccidial activity in vivo, revealed that each served as a substrate for the enzymes from E. tenella and E. acervulina. The E. tenella enzyme was the more efficient at the phosphorylation of those compounds. However, only the ethylthio- compound was detectably phosphorylated by the enzyme from E. brunetti. In contrast to the inhibitor specificity, the substrate activities of these nucleosides do not correlate well with their in vivo anticoccidial activity.

Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase.

Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus represents a promising target for rational design of antiparasitic compounds. In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T. gondii was overexpressed in E. coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of TgAK revealed Km values of 1.9 microM for adenosine and 54.4 microM for ATP, with a k(cat) of 26.1 min(-1). Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs. Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T. gondii AK. Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo.

The partial purification and characterization of adenosine kinase from Entamoeba histolytica.

Axenically grown Entamoeba histolytica was found to contain adenosine kinase. This organism lacks de novo purine biosynthetic pathways. Adenosine kinase provides the amoeba with a method for salvaging adenosine from ingested nucleosides or from degraded nucleotides. Adenosine kinase was purified 64-fold, by chromatography on Sephacryl S-200, DEAE-cellulose, and (C-8)-adenosine-agarose. The latter separated it from amebal adenylate kinase. Adenosine kinase has a molecular weight of 38,000 and requires glycerol for stability. It utilizes adenosine triphosphate to phosphorylate adenosine, and 7-deazaadenosine (tubercidin), but adenine 9-beta-D-arabinofuranoside (ara-A) is not detectably phosphorylated. It requires Mg++ as a cofactor.

Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity.

In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot analysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with beta-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney > spleen > lung > heart > brain > muscle. Measurement of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.

Human placental adenosine kinase: purification and characterization.

Human placental adenosine kinase has thus been purified 3600-fold and characterized with respect to molecular weight, substrate specificity, divalent cation requirements, pH optimum, isoelectric pH, and kinetic properties. These data contribute to the information currently available about the regulation of adenosine metabolism, information critical for an understanding of the biological properties of adenosine.

The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.

AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.

Expression, purification, and characterization of recombinant Saccharomyces cerevisiae adenosine kinase.

Adenosine kinase (AK), a key enzyme in the regulation of the cellular concentrations of adenosine (A), is an important physiological effector of many cells and tissues. In this article, we reported that ak, which encoded adenosine kinase, was cloned from Saccharomyces cerevisiae, sequenced, and overexpressed in E. coli using the pET16b expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of S. cerevisiae AK revealed K(m) values of (3.5+/-0.2) micromol/L for adenosine and (100.0+/-11.0) micromol/L for ATP, with k(cat) of (1530+/-20) min(-1) for adenosine and (1448+/-25) min(-1) for ATP. The determination of the K(m) value for other nucleosides and deoxynucleoside indicated that the nucleoside specificity of this enzyme from yeast was quite high.

Pentavalent ions dependency of mammalian adenosine kinase.

The enzyme adenosine kinase (AK) has been purified to homogeneity from Syrian hamster and bovine livers. The purified enzymes from both these sources have a Mr of approximately 38 kDa, as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis. A novel characteristic of AK observed here is that its catalytic activity shows a nearly complete dependence upon the presence of pentavalent ions such as phosphate (P(i)), arsenate or vanadate. Maximal AK activity was observed in the presence of either 2-3 mM P(i), or 5-10 mM arsenate, or 10-20 mM vanadate. A low basal level of AK activity (1-5% of maximal) observed in the absence of these ions is attributed to P(i) contamination in the adenine nucleotides preparations. The presence of P(i) had no effect on the K m for ATP (0.4 mM), but it markedly increased the affinity of the enzyme for adenosine. The K(m) of AK for adenosine in presence of 0, 0.1 mM and 2 mM P(i) was estimated to be 1.4 mu M, 0.77 mu M and 0.095 mu M, respectively. Free P(i) showed no exchange with any of the reactants during the assay conditions, and its presence had no effect on the thermostability of the enzyme. These observations suggest that the pentavalent ions such as phosphate may be playing an important role in the enzyme's catalytic mechanism by facilitating either binding of adenosine to the enzyme or in the formation of an enzyme-ATP-adenosine complex.

Presence of adenine phosphoribosyltransferase and adenosine kinase in chloroplasts of spinach leaves.

Activity of adenine phosphoribosyltransferase and adenosine kinase was detected in purified spinach chloroplasts by using differential centrifugation and discontinuous Percoll density gradients. This is the first report of purine salvage enzymes being located in chloroplasts. The role of adenine and adenosine salvage in chloroplasts is discussed.

Analysis of adenosine kinase mutants of baby hamster kidney cells using affinity-purified antibody.

The adenosine kinase enzymes from arabinofuranosyladenine-resistant (araAr) mutants of the baby hamster kidney cell line were analyzed using adenosine kinase-specific antibody probes purified by adenosine kinase-Sepharose column chromatography. Wild-type baby hamster kidney cells were shown to produce two adenosine kinase polypeptides of Mr 43,000 and 40,000. The class I araAr mutants that have no detectable adenosine kinase activity are completely deficient in the two adenosine kinase polypeptides. As expected, the class II araAr mutants, which had been shown to have an altered ribonucleotide diphosphate reductase activity, produce a wild-type level of the two adenosine kinase polypeptides. The five class III araAr mutants which are adenosine-sensitive (AdoS) have various levels of adenosine kinase activity and produced two adenosine kinase polypeptides with similar Mr as that of wild-type cells. The adenosine kinase proteins synthesized by two of the AdoS mutants, ara-19a and ara-74b, differed from wild type in their isoelectric points. These results plus the observations that the AdoS mutants produce adenosine kinase enzymes with altered kinetic properties suggest a point mutation in the adenosine kinase gene. An araAr mutant, ara-60a, with intermediate adenosine sensitivity, was shown to have two truncated adenosine kinase polypeptides. This observation strongly supports the genetic data which suggests that there is only one functional adenosine kinase allele in baby hamster kidney cells and that the two adenosine kinase polypeptides are due to posttranscriptional modification.

Isolation and characterization of adenosine kinase from Leishmania donovani.

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified 3250-fold from Leishmania donovani promastigotes using ion-exchange, gel filtration, and affinity chromatography techniques. Both native and sodium dodecyl sulfate-gel electrophoresis of the enzyme revealed a single polypeptide of around 38,000 molecular weight. Biophysical and biochemical analyses of the enzyme reveal unique characteristics different from those of adenosine kinases from other eukaryotic sources. The isoelectric pH of the enzyme is 8.8. In native acrylamide gels the enzyme moves with an RF of about 0.62. The enzyme displays a maximum activity at pH between 7.5 and 8.5 and is dependent upon an optimum ATP/Mg2+ ratio. ATP at high concentration inhibits the reaction. Adenosine and Mg2+ are not inhibitory. EDTA completely knocks off the activity. Enzyme activity is dependent upon the presence of active thiol group(s) at or near the active center. Under a defined set of conditions the enzyme exhibited an apparent Km for adenosine and ATP of 33 and 50 microM, respectively. Of the nucleoside triphosphates tested ATP and GTP were the most effective phosphate donors. Marginal inhibition of activity was detected with other nucleosides as competitors. However, adenosine analogs, such as 7-deaza-adenosine (tubercidin) and 6-methylmercaptopurine riboside at very low concentrations, were found to be excellent inhibitors and substrates as well. S-Adenosylhomocysteine does not inhibit the reaction even at very high concentration.

Isolation and immunological properties of adenosine kinase.

Bovine liver adenosine kinase is a 43 kDa protein that catalyzes the transfer of phosphate from GTP or ATP to adenosine. Its immunological properties were compared to other GTP-binding proteins of approximately 40 kDa, in particular those involved in signal transduction, such as Gs and Gi, the stimulatory and inhibitory regulatory proteins of adenylyl cyclase, Gt, from the visual excitation system, and Go, a similar protein of unknown function. Antibodies elicited in rabbits against adenosine kinase did not significantly cross-react with other guanyl nucleotide-binding proteins. Antibodies against the other GTP-binding proteins did not react with adenosine kinase. Thus these GTP-binding proteins do not exhibit immunological cross-reactivity.

A novel human phosphotransferase highly specific for adenosine.

A novel nucleoside phosphotransferase, referred to as adenosine phosphotransferase (Ado Ptase), was partially purified 1230-fold from human placenta. This enzyme differed from other known nucleoside phosphotransferases in its substrate specificity. Using AMP as the phosphate donor, it readily phosphorylated Ado. Changes in the sugar moiety were tolerated. dAdo and ddAdo were phosphate acceptors and dAMP was a donor. No other nucleotide or nucleoside common in nature displayed appreciable activity as donor or acceptor substrate, respectively. In the absence of nucleoside, the enzyme catalyzed the hydrolysis of AMP, typical of other nucleoside phosphotransferases. However, in the presence of Ado, little, if any, hydrolysis occurred. Ado Ptase had an absolute requirement for a metal cation, with Mg2+ and, to a lesser extent, Mn2+ fulfilling this requisite. The apparent Km for Ado was 0.2 mM. However, the donor AMP displayed cooperativity in both transfer and hydrolytic reactions. This cooperativity was eliminated by nucleotides, 2,3-diphosphoglycerate, and inorganic phosphate. ADP and 2,3-diphosphoglycerate were especially potent. In the presence of these effectors, the apparent Km for AMP was 3.0 mM in the transfer reaction and 4.0 mM in the hydrolytic reaction. Kinetic data suggest that there are two nucleotide binding sites on Ado Ptase, one for the donor, the other for an effector. AMP appeared to bind to both sites. Although this novel enzyme might play a role in the anabolism of nucleoside analogues, the normal physiological role of this nucleoside phosphotransferase is not understood.