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.
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.
Vessey
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DA
Liver Study Unit, Department of Veterans' Affairs Medical Center, San Francisco, CA 94121, USA.
Kelley
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Lau
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes / Q000378:metabolism; D002417:Cattle; D003066:Coenzyme A Ligases / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods*; D013379:Substrate Specificity; D015262:Xenobiotics / Q000737:chemistry*
D002250:Carbon Radioisotopes; D011756:Diphosphates; D010761:Phosphorus Radioisotopes; D015262:Xenobiotics; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C107128:xenobiotic medium chain fatty acid - coenzyme A ligase