Study on Membrane Function by the Mutants With Requirement for Phosphatidylinositol.

磷脂酰肌醇（PI）是真核细胞的主要膜脂之一。本研究以C_3H/He小鼠自发性乳腺癌细胞系FM_（3A）细胞为材料，研究了磷脂酰肌醇（myo-inositol，Ins）在合成和组装生物膜中的生理功能，发现该细胞系生长需要10 μ M以上的肌醇（Ins）。在PI存在下生长的小鼠FM 3A细胞中，代谢标记实验和细胞磷脂的定量分析显示：1）PI含量增加，2）磷酸肌醇合成刺激，3）磷脂酰甘油（PG）含量降低，4）[^3H]Ins掺入脂质组分刺激。[^3H]标记的PI掺入细胞脂质组分中，不受预培养过程中Ins浓度和胎牛血清种类的影响。合并[^3H] 关于我们 在约中性pH（pH7.4）和低Ins浓度（11 μ M）下测定这些匀浆的Ins进入脂质级分（PI合成活性）。[^3H]Ins不依赖于CDP-甘油二酯掺入脂质组分（PIE）的量是依赖于CDP-甘油二酯掺入脂质组分（PIS）的量的1.5倍。随着PI的加入，PIS几乎被抑制，PIE与补充PI的浓度成比例地增加。在一个需要PI的突变体（6-2-III）中，[^3H]Ins掺入细胞或细胞脂质部分分别减少到40%，60-70%。突变体比野生型的世代时间长1.5 - 2倍，细胞聚集性降低。在另一个突变体（17-6-I）中，即使在不含Ins的培养基中，PI的加入也能维持较高的生长速率，[^3H]PI掺入细胞脂质组分的量增加了约1.4倍。结果表明：（1）PI对细胞PI的调控，PI：Ins交换酶活性对PI含量增加的贡献，以及PI营养缺陷型突变体中Ins或PI掺入细胞或脂质组分的改变，对阐明PI和Ins在真核生物膜中的生理功能具有重要意义。少

Phosphatidylinositol (PI) is one of the major membrane lipids in eukaryotic cells. Its phosphorylated or degraded derivatives are now well known to be involved in important signal transduction pathways.The physiological function of phosphatidylinositol involving synthesis and assembly of bio-membrane was examined with mouse FM3A cells, derived from spontaneous mammary carcinoma of C3H/He mouse FM3A cells.Above 10 uM of myo-inositol (Ins) was required for growth of this cell line. The metabolic label experiments and quatitative analysis of cellular phospholipids in mouse FM3A cells grown in the presence of PI showed 1) increase of PI content, 2) stimulation of phosphoinositides synthesis, 3) decrease of phosphatidylglycerol (PG) content, and 4) stimulation of [^3H]Ins incorporation into lipid fractions. [^3H]labeled PI was incorporated into cellular lipid fractions without influence with the concentration of Ins or the kinds of fetal calf serum during pre-culture. Incorporation of [^3H] … More Ins into lipid fraction by these homogenates (PI synthesizing activity) was assayed at about neutral pH (pH 7.4) and the low concentration of Ins (11 uM). CDP-diacylglycerol independent incorporation of [^3H]Ins into lipid fraction (PIE) was 1.5 fold greater than CDP-diacylglycerol dependent one (PIS). With addition of PI, PIS was almost inhibited, and PIE was increased in proportion to the concentration of supplemented PI. In one of the mutants with requirement for PI on the agar plate (6-2-III), incorporation of [^3H]Ins into cells or cellular lipid fraction reduced to 40 %, 60-70% respectively. The mutant revealed the longer generation time (1.5 - 2 fold) and decrease of cell aggregation than the wild type's. In another mutant (17-6-I), even in the medium without Ins, the addition of PI could maintained the higher growth rate and incorporation of [^3H]PI into cellular lipid fraction was increased about 1.4 fold. The results that (1) the manipulation of cellular PI by the medium supplemented with PI, contribution of PI : Ins exchange enzyme activity to the increase of content of cellular PI and alteration of Ins or PI incorporation into cells or lipid fraction in PI auxotrophic mutants suggested the important consequences to clarify the physiological function of PI and Ins in eukaryotic biomembrane. Less