Adenylate deaminase deficiency in a mutant murine T cell lymphoma cell line.
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From a population of wild type S49 cells, a clone, DTB6, was isolated in a single step from selective medium containing thymidine and dibutyryl cyclic AMP that exhibited a 60% deficiency in AMP deaminase (AMP-D) activity. The AMP-D deficiency conferred to the DTB6 cells a striking susceptibility to killing by low concentrations of either adenine or adenosine, the latter in the presence of an inhibitor of adenosine deaminase activity. This growth supersensitivity of DTB6 cells toward adenine could be ameliorated by the addition of hypoxanthine to the culture medium. Immunoprecipitation of AMP-D from wild type and mutant cells revealed that the DTB6 cell line contained markedly diminished amounts of the AMP-D isozyme which reacts with antisera to the predominant isoform expressed in adult kidney. The quantities of the AMP-D isozyme immunoprecipitated by antisera raised to the predominant isoform prepared from adult heart were equivalent in the two cell lines. Although Northern blot analyses revealed no alterations in mRNA sizes or levels encoded by either of the AMP-D genes, Southern blots of genomic DNA hybridized to a cDNA specific for the ampd2 gene revealed the presence of a new BamHI restriction fragment in the DNA of DTB6 cells. These data suggested that a point mutation has occurred in the ampd2 gene of DTB6 cells which encodes the AMP-D isozyme recognized by the kidney antisera. The DTB6 cells also possessed a virtual complete deficiency in thymidine kinase activity. The two enzyme deficiencies were distinguishable. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism. (+info)
Adenylate deaminase. A multigene family in humans and rats.
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Multiple AMP deaminase (AMP-D) isoforms have been found in vertebrates, and tissue-specific inherited deficiencies of AMP-D have been described in two unrelated clinical syndromes suggesting there may be more than one AMP-D gene in higher eukaryotes. Using a newly isolated cDNA cloned from an adult rat brain library and a previously reported cDNA cloned from adult rat skeletal muscle, two linked AMP-D genes have been identified in rat and man. ampd1 is expressed at high levels in skeletal muscle of the adult rat. ampd2 is the predominant gene expressed in non-muscle tissues and smooth muscle of the adult rat, and it is also the predominant gene expressed in embryonic muscle and undifferentiated myoblasts. Both genes are expressed in cardiac muscle of the adult rat. The peptides encoded by these two genes have distinct immunological properties. The conservation of nucleotide sequence and exon/intron boundaries in these two genes suggests they arose by duplication of a common primordial gene around 150 million years ago. (+info)
Structure of diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino)uracil reductase from Acinetobacter baumannii.
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A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPD1.
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AMP deaminase (AMPD) is a central enzyme in eucaryotic energy metabolism, and tissue-specific as well as stage-specific isoforms are found in many vertebrates. This study demonstrates the AMPD1 gene product in rat is alternatively spliced. The second exon, a 12-base miniexon, was found to be excluded or included in a tissue-specific and stage-specific pattern. This example of cassette splicing utilizes a unique pathway through an RNA intermediate that generates an alternative 5' splice donor site at the point where exon 2 is ligated to exon 1. In the analogous intermediate of human AMPD1, the potential 5' splice donor site created at the boundary of exon 1 and exon 2 was a poor substrate for splicing because of differences in exon 2 sequences, and human AMPD1 was not alternatively spliced. These results demonstrate that in some cases alternative splicing may proceed through an RNA intermediate that generates an alternative splice donor site not present in the primary transcript. Discrimination between alternative 5' splice donor sites in the RNA intermediate of AMPD1 is apparently controlled by tissue-specific and stage-specific signals. (+info)
Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism.
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Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast. (+info)
Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5'-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.
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With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5'-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements producing the cytotoxic effect of this drug by (1) producing tighter binding of methotrexate to folate-dependent enzymes, (2) producing inhibitors of folate-dependent enzymes from their tetrahydrofolate coenzymes, and (3) trapping toxic amounts of adenine nucleosides and nucleotides as a result of inhibition of adenosine deaminase and 5'-adenylate deaminase respectively. (+info)
Identification of a 5'-deoxyadenosine deaminase in Methanocaldococcus jannaschii and its possible role in recycling the radical S-adenosylmethionine enzyme reaction product 5'-deoxyadenosine.
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Expression of three stage-specific transcripts of AMP deaminase during myogenesis.
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AMP deaminase, a ubiquitous enzyme in eucaryotes, plays a central role in energy metabolism. In the present study, RNase protection analyses and immunoprecipitation with tissue-specific antisera were used to examine the transcripts and peptides of AMP deaminase produced during myogenesis in vivo and during myocyte differentiation in vitro. In embryonic muscle and undifferentiated myoblasts, a 3.4-kilobase (kb) transcript encoded a 78-kilodalton (kDa) AMP deaminase peptide that cross-reacted with antisera raised to the AMP deaminase isoform purified from kidney of the adult animal. In perinatal muscle and myocytes at an intermediate stage of differentiation in vitro, a 2.5-kb transcript was produced, and it encoded a 77.5-kDa AMP deaminase peptide that cross-reacted with antisera to the isoform purified from adult heart muscle. At about the time of birth, another 2.5-kb AMP deaminase transcript that encoded an 80-kDa peptide became detectable. This peptide cross-reacted with antisera to the predominant isoform purified from adult skeletal muscle. (+info)