Dephospho-CoA Kinase Provides a Rapid and Sensitive Radiochemical Assay for Coenzyme A and Its Thioesters
| Autor: | John E. Cronan, Caryn S. Wadler |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2007 |
| Předmět: |
chemistry.chemical_classification
Chromatography Coenzyme A Phosphatase Acetyl-CoA Biophysics Esters Cell Biology Biochemistry Article chemistry.chemical_compound Phosphotransferases (Alcohol Group Acceptor) Enzyme Malonyl-CoA chemistry Alkaline phosphatase Scintillation Counting Succinyl-CoA Biological Assay Acyl Coenzyme A Trichloroacetic acid Molecular Biology Chromatography High Pressure Liquid |
| Popis: | 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 [gamma-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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|