Noy N, Zakim D
Biochemistry. 1985 Jul 2;24(14):3521-5. doi: 10.1021/bi00335a020.
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 data indicate, therefore, that the activity of this enzyme 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. It is shown too that the rate of spontaneous transfer of palmitate from unilamellar vesicles of phosphatidylcholine to microsomes via a hydrated intermediate is far more rapid than the inherent catalytic activity of the fatty acyl-CoA ligase. The data also suggest that the membrane-associated pool of fatty acid and not fatty acid in the aqueous phase of the assay is the pool of substrate interacting with the ligase.
将掺入磷脂酰胆碱单层囊泡中的棕榈酸盐用作大鼠肝脏微粒体中棕榈酰辅酶A连接酶（棕榈酰辅酶A连接酶）的底物。这样做是为了避免使用洗涤剂来分散水不溶性棕榈酸盐，以及避免将作为盐悬浮液添加到水性测定中的棕榈酸盐沉淀的可能性。当将棕榈酸盐作为磷脂囊泡的组分添加到测定中时，所测得的连接酶活性比文献中使用其他方法将脂肪酸添加到测定系统中所报道的活性高10至40倍。然而，磷脂对棕榈酰辅酶A连接酶的活性没有直接影响。因此，数据表明，由于脂肪酸添加到测定中的方式对连接酶的活性有显著影响，该酶的活性被低估了。还表明，棕榈酸盐通过水合中间体从磷脂酰胆碱单层囊泡自发转移到微粒体的速率比脂肪酰辅酶A连接酶的固有催化活性快得多。数据还表明，与连接酶相互作用的底物池是与膜相关的脂肪酸池，而不是测定水相中的脂肪酸池。
