Momchilova A, Ivanova L, Markovska T, Pankov R
Institute of Biophysics, Bulgarian Academy of Sciences, Sofia.
Arch Biochem Biophys. 2000 Sep 15;381(2):295-301. doi: 10.1006/abbi.2000.1986.
The content of phosphatidylserine (PS) was found to be increased three times in the plasma membrane outer leaflet of ras-transformed fibroblasts compared to their nontransformed counterparts. In an attempt to determine the mechanisms responsible for the enhanced external appearance of PS, we investigated the activities of aminophospholipid translocase and the nonspecific lipid scramblase. Both transport systems could separately or in combination contribute to PS accumulation in the extracellular leaflet. Aminophospholipid transfer was assessed by measuring the rate of NBD-PS internalization, and scramblase activity was estimated from the internalization of NBD-PC. The results showed that the aminophospholipid transport was inhibited and the nonspecific transport was stimulated in ras-transformed cells. To assess which of these two transport systems was related to elevation of PS external appearance, each of them was submitted to reversible alterations and the content of PS was measured simultaneously. Aminophospholipid translocase activity was inhibited by pyridyldithioethylamine treatment and reversed by reduction with dithiothreitol. Scramblase activity was modulated by a calcium repletion-depletion procedure. Calcium depletion was performed by cell incubation with BAPTA-AM and EGTA as Ca2+ intracellular and extracellular chelators. Restoration of the intracellular Ca2+ was achieved by cell incubation with Ca2+ and Ca2+-ionophore A23187. The results showed that the changes in PS outer appearance did not correlate with the uptake of NBD-PS but were closely related to NBD-PC internalization, suggesting that the nonspecific bidirectional lipid transfer was the major transport system translocating PS to the outer leaflet in ras-transformed cells.
与未转化的成纤维细胞相比，ras转化的成纤维细胞质膜外小叶中的磷脂酰丝氨酸（PS）含量增加了两倍。为了确定导致PS在细胞外出现增加的机制，我们研究了氨基磷脂转位酶和非特异性脂质翻转酶的活性。这两种转运系统单独或共同作用都可能导致PS在细胞外小叶中的积累。通过测量NBD-PS内化速率来评估氨基磷脂转移，通过NBD-PC的内化来估计翻转酶活性。结果表明，在ras转化的细胞中，氨基磷脂转运受到抑制，而非特异性转运受到刺激。为了评估这两种转运系统中的哪一种与PS在细胞外出现增加有关，我们对它们分别进行了可逆性改变，并同时测量了PS的含量。氨基磷脂转位酶活性通过吡啶二硫代乙胺处理受到抑制，并通过二硫苏糖醇还原而逆转。翻转酶活性通过钙补充-耗尽程序进行调节。通过用BAPTA-AM和EGTA作为细胞内和细胞外Ca2+螯合剂孵育细胞来实现钙耗尽。通过用Ca2+和Ca2+离子载体A23187孵育细胞来实现细胞内Ca2+的恢复。结果表明，PS在细胞外出现的变化与NBD-PS的摄取无关，但与NBD-PC的内化密切相关，这表明非特异性双向脂质转移是ras转化细胞中将PS转运到外小叶的主要转运系统。
