[Abstract]  [Full Text]  [PDF]

David H. Keating¹, Ana Shulla¹, Adam H. Klein¹ and Alan J. Wolfe^1*

¹ Department of Microbiology and Immunology Loyola University Chicago.

* To whom correspondence should be addressed: Alan J. Wolfe, Department of Microbiology and Immunology Loyola University Chicago. 2160 S. First Ave. Bldg. 105 Maywood, IL 60153.. USA. Phone: (708) 216 5814. Fax: (708) 216 9514. Email: awolfe@lumc.edu

Biol. Proced. Online 2008;10:36-46. doi:10.1251/bpo141
Submitted: July 25, 2007; Accepted: December 13, 2007; Published: March 03, 2008.

Indexing terms: Phosphoric Acid Esters; Chromatography, Thin Layer.

Abbreviations: 2D-TLC, two-dimensional thin layer chromatography; acetyl-P, acetyl phosphate; PTA, phosphotransacetylase; PDHC, pyruvate dehydrogenase complex.

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.