Dataset Information

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)c 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 10-100-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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Skeletal Muscle, Liver

SUBMITTER: Jacek Wisniewski

LAB HEAD: Jacek R Wisniewski

PROVIDER: PXD002156 | Pride | 2015-06-22

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20120523_EXQ4_JRW_SA_94_T_1.raw	Raw
	20120523_EXQ4_JRW_SA_94_T_2.raw	Raw
	20120523_EXQ4_JRW_SA_94_T_3.raw	Raw
	20120523_EXQ4_JRW_SA_95_L_1.raw	Raw
	20120523_EXQ4_JRW_SA_95_L_2.raw	Raw

Items per page:

1 - 5 of 19

Publications

Integrating Proteomics and Enzyme Kinetics Reveals Tissue-Specific Types of the Glycolytic and Gluconeogenic Pathways.

Wiśniewski Jacek R JR Gizak Agnieszka A Rakus Dariusz D Rakus Dariusz D

Journal of proteome research 20150701 8

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 g ...[more]

PMID: 26080680

Similar Datasets

Project description:Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. The bacterium Sinorhizobium meliloti has a NAD(P)-malic enzyme (DME) and a NADP-malic enzyme (TME) and DME is required for symbiotic N2-fixation. To help understand the role of these enzymes, we examined growth, metabolic and transcriptional consequences resulting from the deletion of these enzymes. Few effects were observed upon growth with glucose, whereas growth with the gluconeogenic substrate, succinate, resulted in transcriptional and metabolic effects particularly in the dme mutant strains. When grown with succinate, DME mutant cells accumulated hexose sugar phosphates and trehalose, while TME mutants accumulated putrescine. Succinate-grown DME mutant cells also showed increased transcription of genes for gluconeogenesis and for pathways such as amino acid and fatty acid synthesis that divert metabolites away from the TCA cycle. These data suggested that, DME is required to regulate the levels of TCA cycle intermediates and that the activity of TME is insufficient to prevent the accumulation of TCA cycle intermediates in cells utilizing succinate as carbon source. Consistent with this, in short-1-3 hour incubations with succinate, dme mutant cells excreted large amounts of malate whereas little malate was excreted from tme or wild-type cells. These results support the suggestion that DME is required for N2-fixation in alfalfa because it is required for synthesis of pyruvate and acetyl-CoA and the rapid metabolism of C4-dicarboxylates supplied by the plant. RNA expression was measured for S. meliloti in exponential phase grown in MOPS (morpholinpropanesulfonic acid) buffered minimal media under 2 growth conditions: 1) 15 mM succinate, 2) 15 mM glucose; using wild type cells as well as dme and tme mutant strains (6 experiments, 2 replicates: total 12 samples)