title: Quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis.
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.
authors.affiliation: From the Department of Chemistry.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Metabolomics Facility, Center for Molecular Toxicology and Carcinogenesis, and.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Chiaro
authors.affiliation: Metabolomics Facility, Center for Molecular Toxicology and Carcinogenesis, and the CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences (CAS), Wuhan 430071, China, and.
authors.forename: Limin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Metabolomics Facility, Center for Molecular Toxicology and Carcinogenesis, and.
authors.forename: Philip B
authors.initials: PB
authors.lastname: Smith
authors.affiliation: Department of Engineering Science and Mechanics, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Chung Yu
authors.initials: CY
authors.lastname: Chan
authors.affiliation: From the Department of Chemistry.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Pedley
authors.affiliation: From the Department of Chemistry.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Pugh
authors.affiliation: the Departments of Biochemistry and Cell Biology and Chemistry, Stony Brook University, Stony Brook, New York 11794.
authors.forename: Jarrod B
authors.initials: JB
authors.lastname: French
authors.affiliation: Metabolomics Facility, Center for Molecular Toxicology and Carcinogenesis, and adp117@psu.edu.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Patterson
authors.affiliation: From the Department of Chemistry, sjb1@psu.edu.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D000265:Adenylosuccinate Synthase / Q000032:analysis / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / Q000032:analysis / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007168:IMP Dehydrogenase / Q000032:analysis / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D055432:Metabolomics / Q000379:methods*; D009097:Multienzyme Complexes / Q000032:analysis / Q000378:metabolism*; D011685:Purine Nucleotides / Q000032:analysis / Q000378:metabolism*; D011687:Purines / Q000032:analysis / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: C492322:2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; D001562:Benzimidazoles; D009097:Multienzyme Complexes; D011685:Purine Nucleotides; D011687:Purines; D007168:IMP Dehydrogenase; D000265:Adenylosuccinate Synthase; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C022385:phosphoribosylformylglycinamidine synthetase; C030985:purine
keywords: Mass Spectrometry (MS); Metabolic Flux; Metabolomics; Nucleoside/Nucleotide Biosynthesis; Protein Complex; Purine; Purinosome