title: Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways.
abstract: Glycolysis is the core metabolic pathway supplying energy to cells. Whereas the vast majority of studies focus on specific aspects of the process, global analyses characterizing simultaneously all enzymes involved in the process are scarce. Here, we demonstrate that quantitative label- and standard-free proteomics allows accurate determination of titers of metabolic enzymes and enables simultaneous measurements of titers and maximal enzymatic activities (Amax) of all glycolytic enzymes and the gluconeogenic fructose 1,6-bisphosphatase in mouse brain, liver and muscle. Despite occurrence of tissue-specific isoenzymes bearing different kinetic properties, the enzyme titers often correlated well with the Amax values. To provide a more general picture of energy metabolism, we analyzed titers of the enzymes in additional 7 mouse organs and in human cells. Across the analyzed samples, we identified two basic profiles: a "fast glucose uptake" one in brain and heart, and a "gluconeogenic rich" one occurring in liver. In skeletal muscles and other organs, we found intermediate profiles. Obtained data highlighted the glucose-flux-limiting role of hexokinase which activity was always 10- to 100-fold lower than the average activity of all other glycolytic enzymes. A parallel determination of enzyme titers and maximal enzymatic activities allowed determination of kcat values without enzyme purification. Results of our in-depth proteomic analysis of the mouse organs did not support the concepts of regulation of glycolysis by lysine acetylation.
authors.affiliation: †Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, D-82152 Martinsried, Germany.
authors.forename: Jacek R
authors.initials: JR
authors.lastname: Wiśniewski
authors.affiliation: ‡Department of Animal Molecular Physiology, Wroclaw University, PL-50205 Wroclaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Gizak
authors.affiliation: ‡Department of Animal Molecular Physiology, Wroclaw University, PL-50205 Wroclaw, Poland.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Rakus
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D018938:Caco-2 Cells; D002853:Chromatography, Liquid; D057075:Enzyme Assays / Q000379:methods; D004798:Enzymes / Q000378:metabolism*; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D005943:Gluconeogenesis; D006019:Glycolysis; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D053858:Metabolic Networks and Pathways; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000201:enzymology; D009928:Organ Specificity; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D004798:Enzymes; D006597:Fructose-Bisphosphatase
keywords: carbohydrate metabolism; energy metabolism; gluconeogenesis; glycolysis; kcat-values; lysine acetylation; quantitative proteomics; “filter aided sample preparation” absolute protein quantitation; “total protein approach”