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

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.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Animals
  • Carbon Radioisotopes / metabolism
  • Cattle
  • Coenzyme A Ligases / metabolism*
  • Diphosphates / metabolism
  • Mitochondria, Liver / enzymology*
  • Phosphorus Radioisotopes / metabolism
  • Radioligand Assay / methods*
  • Substrate Specificity
  • Xenobiotics / chemistry*

Substances

  • Carbon Radioisotopes
  • Diphosphates
  • Phosphorus Radioisotopes
  • Xenobiotics
  • Adenosine Triphosphate
  • Coenzyme A Ligases
  • xenobiotic medium chain fatty acid - coenzyme A ligase