STUDIES ON ANIMAL CELL SURFACE PHOSPHATIDYLSERINE-FLIPPASE

翻转酶是催化膜脂跨双层运动（翻转）的膜蛋白。翻转酶的结构尚不清楚。到目前为止，已经报道了几次翻转。其中质膜翻转酶的研究较为深入。该蛋白以atp依赖的方式催化细胞表面磷脂酰丝氨酸的跨双层运动。细胞表面翻转酶的功能尚不清楚。有人提出，质膜翻转酶参与维持质膜脂质不对称和/或内吞作用。以前我们已经提出外源性磷脂酰丝氨酸通过细胞表面翻转酶内化(Kobayashi， T.and Arakawa， Y.(1991) J.CellBiol。, 113235 - 244)。在研究外源性磷脂酰丝氨酸进入细胞内化过程中，我们发现含有不饱和脂肪酸的磷脂酰丝氨酸具有细胞毒性。假设磷脂酰丝氨酸抗性细胞在磷脂酰丝氨酸内化的第一步即质膜上的突变中存在缺陷，我们已经分离出磷脂酰丝氨酸抗性突变体。40%的耐磷脂酰丝氨酸细胞在细胞表面磷脂酰丝氨酸的跨双层运动中存在缺陷。利用这些突变体，我们测量了1)膜脂的不对称性和2)内吞作用的活性。磷脂酰丝氨酸抗性突变体保持脂质不对称。另一方面，用路西法黄和马萝卜过氧化物酶（HRP）测定突变细胞的内吞活性约为野生型CHO细胞的一半。我们的研究结果表明，细胞膜磷脂的翻转参与了内吞作用。

Flippases are membrane proteins which catalyze transbilayr movement (flip-flop) of membrane lipids. Structure of flippases are not well understood. Up to now several flippases have been reported. Among them, plasma membrane flippase has been studied well. This protein catalyzes transbilayr movement of cell surface phosphatidylserine in ATP-dependent manner. The function of cell surface flippase is not known. It has been proposed that plasma membrane flippase is involved in the maintenance of plasma membrane lipid asymmetry and/or endocytosis.Previously we have suggested that exogenous phosphatidylserine is internalized via cell surface flippase (Kobayashi, T.and Arakawa, Y.(1991) J.CellBiol., 113,235-244). In the course of studying the internalization of exogenous phosphatidylserine into cells, we found that phosphatidylserine which has unsaturated fatty acid has cytotoxicity. Assuming that phosphatidylserine-resistant cells are defective in the first step of internalization of phosphatidylserine i.e.flip-flop on the plasma membrane, we have isolated phosphatidylserine-resistant mutants. 40% of phosphatidylserine-resistant cells were defective in transbilayr movement of cell surface phosphatidylserine. Using these mutants, we have measured 1) asymmetry of membrane lipids and 2) activity of endocytosis. Phosphatidylserine-resistant mutants retained lipid asymmetry. On the other hand, endocytosis activity of mutant cells, measured by using lucifer yellow and horse radish peroxidase (HRP), was about half of that of wild type CHO cells. Our results suggest that flip-flop of membrane phospholipids are involved in endocytosis.