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Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis
Journal article   Peer reviewed

Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis

Hong Zhao, Christopher R. Chiaro, Limin Zhang, Philip B. Smith, Chung Yu Chan, Anthony M. Pedley, Raymond J. Pugh, Jarrod B. French, Andrew D. Patterson and Stephen J. Benkovic
The Journal of biological chemistry, Vol.290(11), pp.6705-6713
03/13/2015
DOI: 10.1074/jbc.M114.628701
PMCID: PMC4358094
PMID: 25605736

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Abstract

Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome. Background: Metabolic enzymes have been hypothesized to assemble into complex to respond to cellular metabolism changes. Results:De novo purine biosynthesis increases in purinosome-containing cells. Conclusion: Purine metabolism is adjusted by purinosome assembly. Significance: This study indicates that purinosome is a functional multienzyme complex.
 Mass Spectrometry (MS) Metabolic Flux Metabolomics Nucleoside/Nucleotide Biosynthesis Protein Complex Purine Purinosome

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