Millipore filter assay for long-chain fatty acid:CoASH ligase activity using 3H-labeled coenzyme A.
A novel radiochemical assay for long-chain fatty acid:CoASH ligase activity (AMP) (EC 6.2.1.3) has been developed based on the conversion of [3H]CoASH to long-chain fatty acyl CoA. Fatty acyl [3H]CoA was quantitatively retained on Millipore filters upon filtration of the acidified reaction mixture under conditions where the [3H]CoASH was not retained. The assay was developed using microsomes derived from isolated fat cells as the source of fatty acid:CoASH ligase activity. The assay performed at 25 degrees C for 10 min was linear with added microsomal protein up to 7 mug. The assay was linear with time up to 24 min when 1 mug of protein was employed. Fatty acid:CoASH ligase activity was strongly dependent on ATP and magnesium, was stimulated by Triton WR-1339, and was two- to fivefold dependent on added fatty acid. The filter assay is easier than existing assays based on incorporation of labeled fatty acid and is equally sensitive.
D000818:Animals; D002854:Chromatography, Paper / Q000379:methods; D003065:Coenzyme A; D003066:Coenzyme A Ligases / Q000032:analysis*; D005260:Female; D005374:Filtration; D006898:Hydroxylamines; D008851:Micropore Filters; D008862:Microsomes, Liver / Q000201:enzymology; D010169:Palmitic Acids; D051381:Rats; D014316:Tritium
D006898:Hydroxylamines; D010169:Palmitic Acids; D014316:Tritium; D003066:Coenzyme A Ligases; D003065:Coenzyme A