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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
Author(s) -
Vessey Donald A.,
Kelley Michael,
Lau Eva
Publication year - 1998
Publication title -
journal of biochemical and molecular toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.526
H-Index - 58
eISSN - 1099-0461
pISSN - 1095-6670
DOI - 10.1002/(sici)1099-0461(1998)12:3<151::aid-jbt3>3.0.co;2-k
Subject(s) - carboxylic acid , pyrophosphate , biochemistry , enzyme , substrate (aquarium) , mitochondrion , xenobiotic , chemistry , dna ligase , biology , ecology
A radiolabeled ATP assay was developed for measuring carboxylic acid:CoA ligase activity. The assay was designed to measure the formation of [γ‐ 33 P]pyrophosphate from [γ‐ 33 P]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. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 151–155, 1998