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。

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63560074/
• 关键词: Biomembrane; Phospholipid; Phosphatidylinositol; Cultured Mammalian Cells; Mutant; Myo-Inositol; ミオ・イノシト-ル; ホスファチジルイノシトール; ミオ-イノシトール; 細胞増殖; 培養癌細胞

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。目前已知其磷酸化或降解衍生物参与重要的信号转导途径。以C3H/He小鼠自发性乳腺癌FM3A细胞为材料，研究了磷脂酰肌醇参与生物膜合成和组装的生理功能。该细胞系的生长需要10 μ m以上的肌醇（Ins）。对小鼠FM3A细胞在PI作用下的细胞磷脂进行代谢标记实验和定量分析，结果显示：1)PI含量增加，2)刺激磷酸肌苷合成，3)降低磷脂酰甘油（PG）含量，4)刺激[^3H]Ins掺入脂质组分。[^3H]标记PI掺入细胞脂质组分中，预培养过程中不受Ins浓度或胎牛血清种类的影响。在中性pH （pH 7.4）和低浓度Ins （11 uM）条件下，研究了这些匀浆将[^3H]…More Ins并入脂质部分（PI合成活性）。不依赖于cdp -二酰基甘油的[^3H]蛋白掺入脂质部分（PIE）比依赖于cdp -二酰基甘油的（PIS）多1.5倍。随着PI的添加，PIS几乎被抑制，而PIE随PI添加量的增加而成比例增加。在琼脂板上需要PI的突变体之一（6-2-III）中，[^3H]Ins掺入细胞或细胞脂质部分分别减少到40%和60-70%。与野生型相比，突变体代代时间长（1.5 ~ 2倍），细胞聚集量减少。在另一个突变体（17-6-I）中，即使在没有Ins的培养基中，添加PI也能保持较高的生长速度，[^3H]PI在细胞脂质部分的掺入量增加了约1.4倍。结果表明：(1)添加PI的培养基对细胞PI的操纵，PI: Ins交换酶活性对细胞PI含量增加的贡献，以及PI营养不良突变体中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意义。少

项目成果

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Y. Asai、A. Ohta、I. Shibuya 等人：“通过大肠杆菌中主要外膜脂蛋白的形成缺陷来抑制酸性磷脂缺乏的致死作用”J. Bacteriol.171。

T.Shimokawa,I.Shibuya,H.Matsuzaki et al.: "Alteration of cellular level of phosphatidylinositol by its transport from culture media in mouse FM3A cells." J.Cell.Physiol.

T.Shimokawa、I.Shibuya、H.Matsuzaki 等人：“通过小鼠 FM3A 细胞培养基中的磷脂酰肌醇转运来改变其细胞水平。”

H.Matsuzaki・I.Shibuya et al: J.Cell.Physiol.投稿準備中.

H.Matsuzaki・I.Shibuya 等人：J.Cell.Physiol.准备提交。

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T.Hikiji、I.Shlbuya、A.Ohta 等人：“酿酒酵母中编码磷脂酰丝氨酸合酶的 CHOI 基因的破坏。”

T.Hikiji,I.Shibuya,A.Ohta et al.: "Disruption of CHOI gene encoding phosphatidyl-serine synthase in Saccharomyces cerevisiae." J.Biochem.104. 894-900 (1988)

T.Hikiji、I.Shibuya、A.Ohta 等人：“酿酒酵母中编码磷脂酰丝氨酸合酶的 CHOI 基因的破坏。”