Pomorski T, Herrmann A, Müller P, van Meer G, Burger K
Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Humboldt-Universität zu Berlin, Germany.
Biochemistry. 1999 Jan 5;38(1):142-50. doi: 10.1021/bi981244n.
The translocation of spin-labeled analogues of phosphatidylcholine (4-doxylpentanoyl-PC, SL-PC), phosphatidylethanolamine (SL-PE), phosphatidylserine (SL-PS), and sphingomyelin (SL-SM) from the outer to the inner leaflet of the plasma membrane bilayer was investigated in dog kidney MDCK II and human colon Caco-2 cells. Disappearance from the outer leaflet was assayed using back-exchange to serum albumin. Experiments with cells in suspension as well as with polarized cells on filters were performed at reduced temperatures (10 and 20 degreesC) to suppress endocytosis and hydrolysis of spin-labeled lipids. For both epithelial cell lines, a fast ATP-dependent inward movement of the aminophospholipids SL-PS and SL-PE was found, while SL-SM was only slowly internalized without any effect of ATP depletion. The kinetics of redistribution of SL-PC were clearly different between the two cell lines. In MDCK II cells, SL-PC was rapidly internalized in an ATP-dependent and N-ethylmaleimide-sensitive manner and at a rate similar to that of the aminophospholipids. In contrast, in Caco-2 cells the inward movement of SL-PC was much slower than that of the aminophospholipids, did not depend on ATP, and was not N-ethylmaleimide-sensitive. Inhibitor studies indicated that the outward-translocating multidrug resistance P-glycoprotein present in these cells did not affect the kinetics of inward translocation. Internalization was always similar on the apical and basolateral cell surface, suggesting the presence of the same phospholipid translocator(s) on both surface domains of epithelial cells. We propose that Caco-2 cells contain the well-known aminophospholipid translocase, while MDCK II cells contain either two translocases, namely, the aminophospholipid translocase and a phosphatidylcholine-specific translocase, or one translocase of a new type, translocating aminophospholipids as well as phosphatidylcholine.
在犬肾MDCK II细胞和人结肠Caco-2细胞中，研究了磷脂酰胆碱（4-氧代戊酰基-PC，SL-PC）、磷脂酰乙醇胺（SL-PE）、磷脂酰丝氨酸（SL-PS）和鞘磷脂（SL-SM）的自旋标记类似物从质膜双层的外层向内层的转位。使用与血清白蛋白的反向交换来测定外层的消失情况。在降低的温度（10和20℃）下对悬浮细胞以及滤膜上的极化细胞进行实验，以抑制自旋标记脂质的内吞作用和水解。对于这两种上皮细胞系，发现氨基磷脂SL-PS和SL-PE有快速的ATP依赖性向内移动，而SL-SM仅缓慢内化，且不受ATP消耗的影响。两种细胞系中SL-PC重新分布的动力学明显不同。在MDCK II细胞中，SL-PC以ATP依赖性和N-乙基马来酰亚胺敏感的方式快速内化，速率与氨基磷脂相似。相比之下，在Caco-2细胞中，SL-PC的向内移动比氨基磷脂慢得多，不依赖于ATP，且对N-乙基马来酰亚胺不敏感。抑制剂研究表明，这些细胞中存在的向外转运的多药耐药P-糖蛋白不影响向内转位的动力学。顶端和基底外侧细胞表面的内化情况总是相似的，这表明上皮细胞的两个表面结构域存在相同的磷脂转运体。我们提出，Caco-2细胞含有众所周知的氨基磷脂转位酶，而MDCK II细胞要么含有两种转位酶，即氨基磷脂转位酶和磷脂酰胆碱特异性转位酶，要么含有一种新型转位酶，可转运氨基磷脂和磷脂酰胆碱。
