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

Caryn Wadler, John E. Cronan

Research output: Contribution to journalArticlepeer-review

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 languageEnglish (US)
Pages (from-to)17-23
Number of pages7
JournalAnalytical Biochemistry
Volume368
Issue number1
DOIs
StatePublished - Sep 1 2007

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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