Millipore filter assay for long-chain fatty acid:CoASH ligase activity using 3H-labeled coenzyme A

Abstract

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.