Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters.
Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters.
复制标题
去磷酸辅酶 A 激酶为辅酶 A 及其硫酯提供快速、灵敏的放射化学测定。
DOI：
10.1016/j.ab.2007.05.031
复制
发表时间：
2007
影响因子：
2.9
通讯作者：
Cronan,JohnE
中科院分区：
文献类型：
--
作者：
Wadler,Caryn;Cronan,JohnE
报道了一种测定辅酶A（CoA）和短链酰基CoA硫酯体内库的新方法。三氯乙酸提取释放的代谢产物回收和定量脱磷酸虾碱性磷酸酶处理。去除磷酸酶后，通过用[γ-33 P]ATP加脱磷酸CoA激酶处理，脱磷酸CoA代谢物被定量再磷酸化。然后通过反相高效液相色谱法分离得到的放射性CoA代谢物，并通过闪烁计数法进行定量。由于酶促放射性磷酸化，该测定法对CoA及其短链硫酯具有特异性，并且对这些化合物的亚皮摩尔水平敏感。
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.