Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

Caryn Wadler; John E. Cronan

doi:10.1016/j.ab.2007.05.031

Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

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Abstract

A new approach to determine in vivo pools of coenzyme A (CoA) and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp alkaline phosphatase. Following phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with [γ-³³P]ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radiophosphorylation, the assay is specific for CoA and its short chain thioesters and is sensitive to sub-picomole levels of these compounds.

Original language	English (US)
Pages (from-to)	17-23
Number of pages	7
Journal	Analytical Biochemistry
Volume	368
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2007.05.031
State	Published - Sep 1 2007

Keywords

Acetyl-CoA
CoA
Intracellular pools
Malonyl-CoA
Phosphatase
Succinyl-CoA

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

Online availability

10.1016/j.ab.2007.05.031

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title = "Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters",

abstract = "A new approach to determine in vivo pools of coenzyme A (CoA) and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp alkaline phosphatase. Following phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with [γ-33P]ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radiophosphorylation, the assay is specific for CoA and its short chain thioesters and is sensitive to sub-picomole levels of these compounds.",

keywords = "Acetyl-CoA, CoA, Intracellular pools, Malonyl-CoA, Phosphatase, Succinyl-CoA",

author = "Caryn Wadler and Cronan, {John E.}",

note = "Funding Information: We thank Andrei Osterman for the human DPCK. This work was supported by NIH grant AI15650 from the National Institute of Allergy and Infectious Diseases.",

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doi = "10.1016/j.ab.2007.05.031",

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T1 - Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

AU - Wadler, Caryn

AU - Cronan, John E.

N1 - Funding Information: We thank Andrei Osterman for the human DPCK. This work was supported by NIH grant AI15650 from the National Institute of Allergy and Infectious Diseases.

PY - 2007/9/1

Y1 - 2007/9/1

N2 - A new approach to determine in vivo pools of coenzyme A (CoA) and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp alkaline phosphatase. Following phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with [γ-33P]ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radiophosphorylation, the assay is specific for CoA and its short chain thioesters and is sensitive to sub-picomole levels of these compounds.

AB - A new approach to determine in vivo pools of coenzyme A (CoA) and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp alkaline phosphatase. Following phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with [γ-33P]ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radiophosphorylation, the assay is specific for CoA and its short chain thioesters and is sensitive to sub-picomole levels of these compounds.

KW - Acetyl-CoA

KW - CoA

KW - Intracellular pools

KW - Malonyl-CoA

KW - Phosphatase

KW - Succinyl-CoA

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JO - Analytical Biochemistry

JF - Analytical Biochemistry

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Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

Abstract

Keywords

ASJC Scopus subject areas

Online availability

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