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Development of a radiolabeled ATP assay for carboxylic acid:CoA Ligases and its use in the characterization of the xenobiotic carboxylic acid:CoA ligases of bovine liver mitochondria

✍ Scribed by Donald A. Vessey; Michael Kelley; Eva Lau

Publisher
John Wiley and Sons
Year
1998
Tongue
English
Weight
102 KB
Volume
12
Category
Article
ISSN
1095-6670

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✦ Synopsis

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.

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