caa a22 4500 445324791 CHVBK 20180317142716.0 cr unu---uuuuu 170323e20110401xx s 000 0 eng 10.1007/s10858-011-9481-9 doi (NATIONALLICENCE)springer-10.1007/s10858-011-9481-9 Role of aminotransferases in glutamate metabolism of human erythrocytes [Elektronische Daten] [James Ellinger, Ian Lewis, John Markley] Human erythrocytes require a continual supply of glutamate to support glutathione synthesis, but are unable to transport this amino acid across their cell membrane. Consequently, erythrocytes rely on de novo glutamate biosynthesis from α-ketoglutarate and glutamine to maintain intracellular levels of glutamate. Erythrocytic glutamate biosynthesis is catalyzed by three enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamine aminohydrolase (GA). Although the presence of these enzymes in RBCs has been well documented, the relative contributions of each pathway have not been established. Understanding the relative contributions of each biosynthetic pathway is critical for designing effective therapies for sickle cell disease, hemolytic anemia, pulmonary hypertension, and other glutathione-related disorders. In this study, we use multidimensional 1H-13C nuclear magnetic resonance (NMR) spectroscopy and multiple reaction mode mass spectrometry (MRM-MS) to measure the kinetics of de novo glutamate biosynthesis via AST, ALT, and GA in intact cells and RBC lysates. We show that up to 89% of the erythrocyte glutamate pool can be derived from ALT and that ALT-derived glutamate is subsequently used for glutathione synthesis. The Author(s), 2011 Metabolic flux analysis nationallicence Glutathione biosynthesis nationallicence Red blood cells nationallicence Ellinger James Department of Biochemistry, University of Wisconsin-Madison, 53706, Madison, WI, USA aut Lewis Ian Department of Biochemistry, University of Wisconsin-Madison, 53706, Madison, WI, USA aut Markley John Department of Biochemistry, University of Wisconsin-Madison, 53706, Madison, WI, USA aut Journal of Biomolecular NMR Springer Netherlands 49/3-4(2011-04-01), 221-229 0925-2738 49:3-4<221 2011 49 10858 https://doi.org/10.1007/s10858-011-9481-9 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9481-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ellinger James Department of Biochemistry, University of Wisconsin-Madison, 53706, Madison, WI, USA aut NATIONALLICENCE 700 1- Lewis Ian Department of Biochemistry, University of Wisconsin-Madison, 53706, Madison, WI, USA aut NATIONALLICENCE 700 1- Markley John Department of Biochemistry, University of Wisconsin-Madison, 53706, Madison, WI, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 49/3-4(2011-04-01), 221-229 0925-2738 49:3-4<221 2011 49 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer