Vessey D A, Kelley M, Lau E
Liver Study Unit, Department of Veterans' Affairs Medical Center, San Francisco, CA 94121, USA.
J Biochem Mol Toxicol. 1998;12(3):151-5. doi: 10.1002/(sici)1099-0461(1998)12:3<151::aid-jbt3>3.0.co;2-k.
A radiolabeled ATP assay was developed for measuring carboxylic acid:CoA ligase activity. The assay was designed to measure the formation of [gamma-33P]pyrophosphate from [gamma-33P]ATP in the course of the reaction. The assay was linear with protein concentration, and rates as low as 1 pmol/min were measurable. Rates determined with this assay were in agreement with rates determined with [14C]carboxylic acids. The assay was used to characterize the substrate specificity of the XL-I, XL-II, and XL-III ligases from bovine liver mitochondria. Forty carboxylic acids were tested for activity. The enzymes differed in their substrate specificities with XL-I and XL-II being the most similar and XL-III having the broadest specificity. This study has uncovered 19 new carboxylic acids that are substrates for these enzymes.
开发了一种放射性标记的ATP测定法来测量羧酸：辅酶A连接酶的活性。该测定法旨在测量反应过程中[γ-33P]ATP形成[γ-33P]焦磷酸的情况。该测定法与蛋白质浓度呈线性关系，可测量低至1 pmol/分钟的速率。用该测定法测定的速率与用[14C]羧酸测定的速率一致。该测定法用于表征来自牛肝线粒体的XL-I、XL-II和XL-III连接酶的底物特异性。测试了40种羧酸的活性。这些酶的底物特异性不同，XL-I和XL-II最相似，XL-III具有最广泛的特异性。这项研究发现了19种新的羧酸是这些酶的底物。
