Abstract
Abstract:Long‐chain fatty acid:CoA ligase (EC 6.2.1.3) has been examined in vitro with brain preparations from 12‐ to 16‐day‐old rats using oleic (18: 1[n‐9]) and cis‐vaccenic (18: 1[n‐7]) acids. A novel assay system, which permits measurement of product as [14C]fatty acyl‐[3H]CoA, [14C]fatty acyl‐CoA, or fatty acyl‐[3H]CoA, was used. With the double‐label assay, reaction conditions were established that gave a molar ratio of [3H]CoA to [14C]fatty acid in the product of 1.06 ± 0.12 (mean ±s.d., n= 14). This indicates that the method is specific for the fatty acid added to the incubation mixture. The assay is sensitive to the formation of approximately 0.02 nmol fatty acyl‐CoA. With oleic acid, specific activity of the ligase was highest in a microsomal fraction (3.91 ± 0.55 nmol oleoyl‐CoA formed/min/mg protein; n= 16). Optimal activity was obtained at concentrations of fatty acid, CoA, ATP and Mg2+ of 50 μM, 50 μM, 10 mm, and 8 mm, respectively. The apparent Kms (approximate) for oleic acid and coenzyme A were 20 and 6 μM, respectively. With cis‐vaccenic acid (18:1[n‐7]), a naturally occurring isomer of oleate, the maximum reaction rate was higher than that with oleate, as were the apparent Kms for both fatty acid (105 μM) and coenzyme A (23 μM).
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