Fatty acids bound to unilamellar lipid vesicles as substrates for microsomal acyl-CoA ligase

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Abstract
Palmitate incorporated into single-layered vesicles of phosphatidylcholine was used as a substrate for palmitoyl coenzyme A ligase (palmitoyl-CoA ligase) in microsomes from rat liver. This was done in order to avoid the use of detergents for dispersal of the water-insoluble palmitate and the possibility of precipitating palmitate added to the aqueous assay as a salt suspension. The activity of the ligase measured when palmitate was added to assays as a component of phospholipid vesicles was 10-40-fold greater vs. activities reported in the literature using other methods for adding fatty acids to the assay system. Phospholipids, however, had no direct effect on the activity of palmitoyl-CoA ligase. The activity of this enzyme apparently has been underestimated because of the manner in which fatty acid was added to the assay, which has a significant effect on the activity of the ligase. The rate of spontaneous transfer of palmitate from unilamellar vesicles of phosphatidylcholine to microsomes via a hydrated intermediate evidently is far more rapid than the inherent catalytic activity of the fatty acyl-CoA ligase. The membrane-associated pool of fatty acid and not fatty acid in the aqueous phase of the assay apparently is the pool of substrate interacting with the ligase.