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

磷脂酰肌醇需要突变体的膜功能研究。

基本信息

批准号：
63560074
负责人：
MATSUZAKI Hiroshi
金额：
$ 1.22万
依托单位：
Saitama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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

磷脂酰肌醇（PI）是真核细胞中主要的膜脂之一，其磷酸化或降解衍生物现已众所周知参与重要的信号转导途径。用小鼠研究了磷脂酰肌醇涉及生物膜合成和组装的生理功能。 FM3A细胞，源自C3H/He小鼠FM3A细胞的自发性乳腺癌。10 uM以上该细胞系的生长需要肌醇 (Ins)。在 PI 存在下生长的小鼠 FM3A 细胞中的细胞磷脂的代谢标记实验和定量分析显示 1) PI 含量增加，2) 刺激磷酸肌醇合成， 3) 磷脂酰甘油 (PG) 含量减少，4) 刺激 [^3H]Ins 掺入脂质组分。 [^3H]标记的 PI 被掺入细胞脂质组分中，而不受预培养期间 Ins 浓度或胎牛血清种类的影响。通过这些匀浆将 [^3H]…更多 Ins 掺入脂质组分中（PI 合成活性）。 ) 在大约中性 pH (pH 7.4) 和低浓度的 Ins (11 uM) 下进行测定，[^3H]Ins 不依赖于脂质组分的掺入。 (PIE)比CDP-二酰基甘油依赖性蛋白(PIS)高1.5倍，添加PI后，PIS几乎被抑制，并且在需要PI的突变体之一中，PIE与补充PI的浓度成比例地增加。琼脂平板 (6-2-III) 中，[^3H]Ins 掺入细胞或细胞脂质部分分别减少至 40%、60-70%。突变体显示。在另一种突变体（17-6-I）中，即使在不含Ins的培养基中，添加PI也能保持较高的生长速率和掺入。进入细胞脂质部分的[^3H]PI增加了约1.4倍。结果是(1)通过添加PI的培养基对细胞PI的操纵，PI的贡献：Ins交换酶活性对含量增加的影响。细胞 PI 的变化以及 PI 营养缺陷型突变体中 Ins 或 PI 掺入细胞或脂质部分的改变表明了澄清 PI 和 Ins 在真核生物膜中的生理功能的重要后果。