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Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules.

Keating DH, Shulla A, Klein AH, Wolfe AJ - Biol Proced Online (2008)

Bottom Line: This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements.We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts.This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Loyola University Chicago, Chicago, IL, USA.

ABSTRACT
Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.

No MeSH data available.


Fig. 1                  The PDHC-PTA-ACKA pathway: Glucose and serine (the preferred sugar and amino acid carbon sources of E. coli) are metabolized to pyruvate. The pyruvate is converted to acetyl-CoA through the action of pyruvate dehydrogenase complex (PDHC). The acetyl-CoA is converted to acetyl-P by phosphotransacetylase (PTA) and the acetyl-P is converted to acetate by acetate kinase (ACKA). HS-CoA, coenzyme A; NAD+ and NADH, oxidized and reduced forms of nicotinamide adenine dinucleotide; Pi, inorganic phosphate.
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f1: Fig. 1 The PDHC-PTA-ACKA pathway: Glucose and serine (the preferred sugar and amino acid carbon sources of E. coli) are metabolized to pyruvate. The pyruvate is converted to acetyl-CoA through the action of pyruvate dehydrogenase complex (PDHC). The acetyl-CoA is converted to acetyl-P by phosphotransacetylase (PTA) and the acetyl-P is converted to acetate by acetate kinase (ACKA). HS-CoA, coenzyme A; NAD+ and NADH, oxidized and reduced forms of nicotinamide adenine dinucleotide; Pi, inorganic phosphate.

Mentions: Acetyl phosphate (acetyl-P) is the high-energy intermediate of the reversible Pta-AckA pathway (Fig. 1). The Pta-AckA pathway inter-converts acetyl-coenzyme A (acetyl-CoA), ADP, and inorganic phosphate (Pi) with acetate, coenzyme A (HS-CoA) and ATP. When functioning in the direction of acetate synthesis, this pathway serves three functions: 1) to incorporate Pi, 2) to recycle coenzyme A (HS-CoA) and 3) to produce ATP. This synthesis of ATP relies on the ability of acetyl-P to store, in the form of its high-energy phosphate bond, about fifty percent more energy than ATP (reviewed by (1)).


Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules.

Keating DH, Shulla A, Klein AH, Wolfe AJ - Biol Proced Online (2008)

Fig. 1                  The PDHC-PTA-ACKA pathway: Glucose and serine (the preferred sugar and amino acid carbon sources of E. coli) are metabolized to pyruvate. The pyruvate is converted to acetyl-CoA through the action of pyruvate dehydrogenase complex (PDHC). The acetyl-CoA is converted to acetyl-P by phosphotransacetylase (PTA) and the acetyl-P is converted to acetate by acetate kinase (ACKA). HS-CoA, coenzyme A; NAD+ and NADH, oxidized and reduced forms of nicotinamide adenine dinucleotide; Pi, inorganic phosphate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2275044&req=5

f1: Fig. 1 The PDHC-PTA-ACKA pathway: Glucose and serine (the preferred sugar and amino acid carbon sources of E. coli) are metabolized to pyruvate. The pyruvate is converted to acetyl-CoA through the action of pyruvate dehydrogenase complex (PDHC). The acetyl-CoA is converted to acetyl-P by phosphotransacetylase (PTA) and the acetyl-P is converted to acetate by acetate kinase (ACKA). HS-CoA, coenzyme A; NAD+ and NADH, oxidized and reduced forms of nicotinamide adenine dinucleotide; Pi, inorganic phosphate.
Mentions: Acetyl phosphate (acetyl-P) is the high-energy intermediate of the reversible Pta-AckA pathway (Fig. 1). The Pta-AckA pathway inter-converts acetyl-coenzyme A (acetyl-CoA), ADP, and inorganic phosphate (Pi) with acetate, coenzyme A (HS-CoA) and ATP. When functioning in the direction of acetate synthesis, this pathway serves three functions: 1) to incorporate Pi, 2) to recycle coenzyme A (HS-CoA) and 3) to produce ATP. This synthesis of ATP relies on the ability of acetyl-P to store, in the form of its high-energy phosphate bond, about fifty percent more energy than ATP (reviewed by (1)).

Bottom Line: This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements.We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts.This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Loyola University Chicago, Chicago, IL, USA.

ABSTRACT
Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.

No MeSH data available.