Studies on the regulatory mechanisms and the biological significance of metabolisms of membrane phospholipids in mammalian cells by using technics of biochemical genetics.

本研究主要是利用生化遗传学技术，以哺乳动物细胞中与膜磷脂代谢有关的变异体为研究对象。1.从CHO(中国仓鼠卵巢)细胞中分离到一株PS(磷脂酰丝氨酸)调节改变的细胞突变株，推测存在PS生物合成调节因子。2.利用PS合酶I缺陷的变异体成功克隆了PS合酶I和PS脱羧酶的基因，该酶在调节PS和PE(磷脂酰乙醇胺)的合成以及细胞的生长和分裂中具有重要意义。3.更远。通过Sindbis病毒对该突变体的感染实验，表明PS和PE在膜融合中的重要性。4.还分离到一个在磷脂酰肌醇合成的重要过程--钠依赖性肌醇转运过程中存在缺陷的CHO突变体，并推测存在两个肌醇转运系统。5.从CHO细胞中分离到一株具有丝氨酸-谷氨酰基转移酶缺陷的温度敏感突变体，该突变株是神经鞘脂脂合成的限速酶，表明鞘脂是哺乳动物细胞生长所必需的。6.还从CHO细胞中分离到1个PG(磷脂酰甘油)合成酶缺陷突变体，并提出PG和心磷脂在细胞生长中的重要作用。7.从J774.1巨噬细胞中分离到一株耐高浓度脂多糖的突变体，发现经脂多糖处理后，花生四烯酸的释放和O_2~-的产生均有缺陷。此外，磷脂酶激活的调节途径可能参与了干扰素-γ对内毒素诱导的O_2~-产生的控制。8.钙调素在胞外三磷酸腺苷调节细胞膜通透性中的作用。在小鼠3T3细胞中，葡萄糖转运蛋白基因GLUT-1被转化生长因子-β表达，GLUT-1蛋白的糖基化发生改变。

This research was performed mainly by using technics of biochemical genetics with variants of mammalian cells concerning metabolisms of membrane phospholipids. 1. A CHO(Chinese hamster ovary)cell mutant with altered regulation of PS(phosphatidylserine)was isolated and the existence of a regulatory factor of PS biosynthesis was suggested. 2. Gene cloning of PS synthase I and PS decarboxylase was succeeded by using a variant defective in PS synthase I, and significance of the enzyme was suggested in regulation of PS and PE(phosphatidylethanolamine)synthesis and in growth and division of cells. 3. Further. importance of PS and PE in the membrane fusion was suggested by the experiment of Sindbis virus infection to this mutant. 4. A CHO variant defective in the Na' dependent inositol transport, which is an important process in phosphatidylinositol synthesis, was also isolated, and the presence of two inositol transport systems was suggested. 5. A temperature sensitive mutant with a defect in serine-parmitoyl transferase, the rate limiting enzyme of the synthesis of sphingolipids, was isolated from CHO cells, and sphingolipids were suggested essential in the mammalian cell growth. 6. A defective mutant of PG(phosphatidylglycerol)synthase was also isolated from CHO cells and importance of PG and cardiolipin in cellular growth was also suggested. 7. A resistant mutant to high concentrations of LPS(lipopolysaccharide)was isolated from J774.1 macrophage cells, and found a defect in both arachidonate release and O_2^- generation after LPS treatment. Further, regulatory pathways from phospholipases activation was suggested to be involved in the control of LPS-induced O_2^- generation by interferon gamma. 8. Importance of calmodulin in control of the membrane permeability with extra-cellular ATP was suggested. In mouse 3T3 cells, the expression of GLUT-1, a glucose transporter gene, by TGF-beta and an altered glycosylation of GLUT-1 protein were suggested.