Studies on the regulatory mechanisms of phospholipid metabolism and membrane functions by using cultured mammalian cell variants defective in phospholipid biosynthesis.

本研究的目的是在培养的哺乳动物细胞中建立磷脂代谢缺陷的变异体，并通过使用这些变异体来阐明磷脂生物合成及其调控的机制，最终探索磷脂在生物合成中的作用和生物膜的功能。我们从中国仓鼠卵巢(CHO)细胞中建立了缺乏磷脂酰丝氨酸(PS)生物合成的变异体，发现PS的生物合成是由磷脂-胆碱交换酶催化的，并指出PS在细胞生长中的重要性。通过获得PS辅酶A(PSA 1-3)，首次发现哺乳动物细胞中存在两种丝氨酸交换酶(酶I和酶II)。此外，酶I的缺陷导致PS含量下降，从而导致细胞死亡。通过分析该变异细胞感染囊膜RNA病毒的情况，阐明了PS在这一过程中的作用。期待这项研究为寻找降低病毒成熟度的程序提供重要信息。同时还分离到了PS生物合成调控机制缺陷的突变体。在亲本菌株中，添加PS对PS合成有明显的抑制作用。然而，在变异细胞中，这种抑制是缺乏的。进一步的调查目前正在进行中。从CHO细胞中分离到一个补充PSA-3细胞PS需求的基因，并建立了该基因与PS生物合成的关系。从磷脂酶A_2活性缺陷的巨噬细胞系中获得了变种，提示至少存在两种磷脂酶A_2。

The aims of this research are to establish variants defective in the phospholipid metabolisms in cultured mammalian cells and, by suing these variants, to clarify the mechanisms of the phospholipid biosynthesis and its regulation and finally to explore the roles of phospholipids in biosynthesis and functions of biological membranes.1. We established the variants lacking biosynthesis of phosphatidylserine (PS) from Chines hamster ovary (CHO) cells, and biosynthesis of PS was shown catalysed by the phospholipid-choline exchange enzymes and the importance of PS in cell growth was also pointed out.2. By obtaining PS auxotroph (PSA 1-3) the existence of two serine exchange enzymes (Enzyme I and Enzyme II) in mammalian cells was found out for the first time. Furthermore, the defect in Enzyme I was shown to induce the decrease in PS content and the consequent death of the cells.3. By analyzing the infection of this variant cells with enveloped RNA virus the functions of PS in this process was clarified. E expect this study gives important informations for finding the procedures to decrease virus maturation.4. The variant defective in the regulatory mechanism of PS biosynthesis was also isolated. In the parent strain PS synthesis was suppressed specifically with PS added to the growth medium. In the variant cells, however, this suppression was lacking. Further investigations are now in progress.5. A gene from CHO cells which complement PS requirement in PSA-3 cells was isolated and the relationship between this gene and the PS biosynthesis was established.6. Variants from macrophage cell line defective in phospholipase A_2 activity were obtained and the existence of at least two phospholipase A_2 was suggested.