Purification of, Generation of Monoclonal Antibodies to, and Mapping of Phosphoribosyl N-Formylglycinamide Amidotransferase

Barnes, Tristan S.; Bleskan, John; Hart, Iris; Walton, K.; Barton, Jeffrey W.; Patterson, David

doi:10.1021/bi00173a031

Cited by 10 publications

(6 citation statements)

References 31 publications

(30 reference statements)

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Section: Discussionmentioning

confidence: 99%

“…In addition we have published evidence that CHO-K1 AdeB mutants produce undetectable levels of FGAMS (phosphoribosylformylglycinamidine) [31], [32]. We have also reported a mutant CHO-K1 cell that overproduces FGAMS [31]. Deng et al [2] recently presented evidence supporting the hypothesis that triGART and FGAMS are core components of the purinosome and that PPAT and FGAMS interact intracellularly, a hypothesis we proposed previously on the basis of somatic cell genetic evidence [33].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: Cellular models of de novo purine biosynthesis deficiency disorders

Duval

Luhrs

Wilkinson

et al. 2013

Molecular Genetics and Metabolism

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Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, renal stones, combined immunodeficiency, and autistic features. The pathogenetic mechanism is unknown for any of these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: Cellular models of de novo purine biosynthesis deficiency disorders

Duval

Luhrs

Wilkinson

et al. 2013

Molecular Genetics and Metabolism

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“…Protein isolations were performed as previously described (Barnes et al, 1994). Protein concentrations were determined using the BioRad Microassay reagent.…”

Section: Methodsmentioning

confidence: 99%

“…Equal amounts (30-40 μg) of total protein were resolved by gel electrophoresis and electroblotted onto nitrocellulose membranes. Immunoblotting and detection were performed essentially as previously described (Barnes et al, 1994; Brodsky et al, 1997) Protein bands were visualized using CDP- Star substrate according to the supplier's protocol (Applied Biosystems). Images of Western blots were captured using a Diana III camera system (Raytest), and analyzed using AIDA Image Analyzer software (Raytest).…”

Section: Methodsmentioning

confidence: 99%

Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis

Knox

Graham

Bleskan

et al. 2009

Gene

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Mutations in several steps of de novo purine synthesis lead to human inborn errors of metabolism often characterized by mental retardation, hypotonia, sensorineural hearing loss, optic atrophy, and other features. In animals, the phosphoribosylglycinamide transformylase (GART) gene encodes a trifunctional protein carrying out 3 steps of de novo purine synthesis, phosphoribosylglycinamide synthase (GARS), phosphoribosylglycinamide transformylase (also abbreviated as GART), and phosphoribosylaminoimidazole synthetase (AIRS) and a smaller protein that contains only the GARS domain of GART as a functional protein. The GART gene is located on human chromosome 21 and is aberrantly regulated and overexpressed in individuals with Down syndrome (DS), and may be involved in the phenotype of DS. The GART activity of GART requires 10-formyltetrahydrofolate and has been a target for anti-cancer drugs. Thus, a considerable amount of information is available about GART, while less is known about the GARS and AIRS domains. Here we demonstrate that the amino acid residue glu75 is essential for the activity of the GARS enzyme and that the gly684 residue is essential for the activity of the AIRS enzyme by analysis of mutations in the Chinese hamster ovary (CHO-K1) cell that require purines for growth. We report the effects of these mutations on mRNA and protein content for GART and GARS. Further, we discuss the likely mechanisms by which mutations inactivating the GART protein might arise in CHO-K1 cells.

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“…Functional FGAM synthetases purified from chicken liver, Salmonella typhimurium and Escherichia coli consist of monomeric polypeptides of 133 kDa, 135 kDa and 141 kDa, respectively (Schendel et al, 1989). Antibodies raised against FGAM synthetase from Chinese hamster ovary cells cross-react with a human protein with a molecular mass of 150 kDa (Barnes et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

The yexA gene product is required for phosphoribosylformylglycinamidine synthetase activity in Bacillus subtilis

Saxild

Nygaard

2000

Microbiology

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The yexA gene encodes an 84 amino acid reading frame ; in Bacillus subtilis it is positioned between the purC and purQ genes of the purine biosynthetic operon. Disruption of yexA resulted in a purine-auxotrophic phenotype. When yexA was expressed in trans it was able to complement a yexA mutation. Growth experiments and enzyme analysis of yexA mutant strains revealed a defective phosphoribosylformylglycinamidine synthetase (FGAM synthetase). In the organisms in which FGAM synthetase has been studied a single polypeptide is responsible for activity. In some organisms two separate genes -in B. subtilis the purL and purQ genes -encode polypeptides with similarity to the N-terminal and the C-terminal region, respectively, of the singlepolypeptide FGAM synthetase. Thus, active FGAM synthetase in B. subtilis requires the yexA gene product in addition to the purL and purQ gene products. Open reading frames with sequence similarity to yexA are found in other Gram-positive organisms, in a cyanobacterium and in methanogenic archaea. The designation purS is proposed for this novel function in purine biosynthesis in B. subtilis.

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Purification of, Generation of Monoclonal Antibodies to, and Mapping of Phosphoribosyl N-Formylglycinamide Amidotransferase

Cited by 10 publications

References 31 publications

Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: Cellular models of de novo purine biosynthesis deficiency disorders

Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: Cellular models of de novo purine biosynthesis deficiency disorders

Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis

The yexA gene product is required for phosphoribosylformylglycinamidine synthetase activity in Bacillus subtilis

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