Samuel D, Paris S, Ailhaud G
Eur J Biochem. 1976 May 1;64(2):583-95. doi: 10.1111/j.1432-1033.1976.tb10338.x.
Cultured heart cells from chick embryo, present as a confluent monolayer in a minimal medium, take up labelled fatty acids (from C6 to C22) bound to serum albumin with a rapid incorporation into neutral and complex lipids (visible at 15 s), while beta-oxidation is delayed (visible at 15 min). Fatty acids enter the cardiac cells through two mechanisms, one involving a readily saturable process, the other resembling passive diffusion. The saturable transport system recognizes fatty acids with a high affinity, Km from 4.5 to 16 muM. The V is from 0.03 to 0.3 nmol of fatty acid incorporated min-1 (10(6) cells)-1. Competitive inhibition occurs between fatty acids while glucose, aminoacids and lactate do not compete with palmitate for the entry into the cells. The analysis of unesterified fatty acids from the usual culture medium and from the cells show a gradient of concentration up to 54-fold; moreover experiments performed in minimal medium at 20 degrees C show that fatty acids accumulate up to 28-fold in the cells. Efflux rates can be measured after loading the cells at 20 degrees C; the curves are bimodal and the pseudo first-order rate constants thus determined are in the order of 0.5 min-1, as opposed to 2.5 to 5.0 min-1 for the passive-diffusion component calculated for the influx rates. Studies on the role of serum albumin at constant and at variable fatty acid-albumin molar ratios indicate that the rate of uptake is controlled by the total fatty acid concentration and not solely by the concentration of unbound fatty acids. No labelled fatty acid was found to be bound to the cytoplasmic fraction supposed to contain the fatty acids-binding protein; our results are in favour of the presence of a specific transport system for fatty acids either at the level of subcellular organelles or more likely at the surface of cardiac cells.
来自鸡胚的培养心肌细胞,在基础培养基中呈汇合单层状态,摄取与血清白蛋白结合的标记脂肪酸(碳链长度从C6到C22),并迅速掺入中性和复合脂质中(15秒时可见),而β-氧化则延迟(15分钟时可见)。脂肪酸通过两种机制进入心肌细胞,一种涉及易饱和过程,另一种类似于被动扩散。可饱和转运系统对脂肪酸具有高亲和力,米氏常数(Km)为4.5至16微摩尔。转运速率(V)为每分钟掺入0.03至0.3纳摩尔脂肪酸(每10^6个细胞)。脂肪酸之间存在竞争性抑制,而葡萄糖、氨基酸和乳酸不与棕榈酸竞争进入细胞。对常规培养基和细胞中未酯化脂肪酸的分析显示,浓度梯度高达54倍;此外,在20℃的基础培养基中进行的实验表明,脂肪酸在细胞中积累高达28倍。在20℃加载细胞后可测量流出速率;曲线呈双峰,由此确定的伪一级速率常数约为0.5分钟^-1,而流入速率计算得出的被动扩散成分的速率常数为2.5至5.0分钟^-1。在脂肪酸-白蛋白摩尔比恒定和可变的情况下对血清白蛋白作用的研究表明,摄取速率由总脂肪酸浓度控制,而不仅仅由未结合脂肪酸的浓度控制。未发现标记脂肪酸与假定含有脂肪酸结合蛋白的细胞质部分结合;我们的结果支持在亚细胞器水平或更可能在心肌细胞表面存在脂肪酸特异性转运系统。