Quantitative Analysis of Purine Nucleotides Indicates Purinosome s Increase de N ovo Purine Biosynthesis

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-dimethylamino4,5,6,7-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 purinedepleted medium) and normal cells showed a three-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP, 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 also were 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. INTRODUCTION Purine nucleotides have very important and diverse functions inside the cell. They are building blocks of DNA and RNA, energy carriers, cell http://www.jbc.org/cgi/doi/10.1074/jbc.M114.628701 The latest version is at JBC Papers in Press. Published on January 20, 2015 as Manuscript M114.628701 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Purinosomes form to meet cellular demands for purines