Morand O, Aigrot M S
Biochim Biophys Acta. 1985 Jun 14;835(1):68-76. doi: 10.1016/0005-2760(85)90031-1.
Human erythrocyte ghosts were used for studying the mechanism of uptake and membrane transport of fatty acids. Hemoglobin-free ghosts were prepared and loaded with substrates such as CoA and/or ATP, and their ability for transporting and activating radiolabelled palmitic acid was tested further. Uptake of radiolabelled palmitic acid by CoA- and ATP-loaded ghosts exceeded that observed with ghosts loaded only with ATP, the latter being greater than that measured with non-loaded ghosts. Acyl-CoA was synthesized in CoA- and ATP-loaded ghosts upon incubation with radiolabelled palmitic acid. Both CoA and ATP were needed within the ghosts to permit acyl-CoA synthesis, suggesting that the acyl-CoA synthetase is located in and is bound to the inner layer of the membrane. The rate of acyl-CoA synthesis was saturable with increasing concentration of palmitic acid in the incubation mixture, and kinetic parameters were calculated. The rate of acyl-CoA synthesis in CoA- and ATP-loaded ghosts upon incubation with radiolabelled palmitic acid was markedly decreased when increasing albumin concentration in the incubation medium up to a molar ratio albumin/fatty acid of one to one. It is not easy to distinguish experimentally fatty acids located in the outer layer and the inner layer of the membrane and the data of this paper suggest that acyl-CoA synthesis by an enzyme located in the inner layer could be used as a measure of the acyl groups which have been translocated across the membrane of erythrocyte ghosts.
人红细胞血影被用于研究脂肪酸的摄取和膜转运机制。制备了无血红蛋白的血影，并装载了诸如辅酶A和/或ATP等底物，进一步测试了它们转运和激活放射性标记棕榈酸的能力。装载辅酶A和ATP的血影对放射性标记棕榈酸的摄取超过了仅装载ATP的血影，而后者又大于未装载的血影。在与放射性标记棕榈酸孵育时，装载辅酶A和ATP的血影中合成了酰基辅酶A。血影内需要辅酶A和ATP才能进行酰基辅酶A的合成，这表明酰基辅酶A合成酶位于膜的内层并与之结合。随着孵育混合物中棕榈酸浓度的增加，酰基辅酶A的合成速率达到饱和，并计算了动力学参数。当孵育培养基中白蛋白浓度增加至白蛋白/脂肪酸摩尔比为1:1时，装载辅酶A和ATP的血影在与放射性标记棕榈酸孵育时酰基辅酶A的合成速率显著降低。通过实验区分位于膜外层和内层的脂肪酸并不容易，本文的数据表明，由位于内层的酶进行的酰基辅酶A合成可作为已转运穿过红细胞血影膜的酰基的一种衡量指标。
