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Study of the mechanism of phospholipase C activation via a new GTP binding protein

通过新型 GTP 结合蛋白激活磷脂酶 C 的机制研究

基本信息

批准号：
62571026
负责人：
NAKAHATA Norimichi
金额：
$ 1.28万
依托单位：
Fukushima Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

This study was undertaken to elucidate the GTP binding protein to activate phospholipase C (PLase C) which is not a substarate for pertussis toxin (IAP) in human astrocytoma cells. The stimulation of TXA_2-receptors as well as muscarinic, H_1-histamine and bradykinin resulted in activation of PLase C in an IAP insensitive manner. The GTP binding protein involved in these agonists-induced PLase C activations is not a substrate for IAP. Pretreatment of cells with agonists elicited the reduction of GTPgammaS-induced accumulation of inositol phosphates (IP) in membrane preparations, reflecting from a functional reduction of signal transduction after agonist treatment of intact cells. Heavy peak fraction of membranes after sucrose gradient separation contained receptors and GTP binding proteins. Receptors together with GTP binding proteins moved from heavy membrane fraction to light peak fraction after treatment of the intact cells with agonists. The reduction of GTP binding protein in heav … More y peak fraction after agonist treatment was analyzed by a photoaffinity labelling with [^<35>S]GTPgammaS. The 32 kDa GTP binding protein was reduced in Heavy peak fraction after treatments of the intact cells with agonists. The 32 kDa GTP binding protein might be involved in receptor-mediated activation of phospholipase C. Then, the 32 kDa GTP binding protein was purified from the porcine brain membranes. There is a 32 kDa GTP binding protein in the membranes, determined by a photoaffinity labelling with [^<32>P]alpha-GTP. The 32 kDa GTP binding protein was solubilized with 1 % lubrol from cholate-inextracted membranes. The 32 kDa gtp binding protein might be hydrophobic, and was not ADP-ribosylated with IAP. The 32 kDa GTP binding protein was purified by column chromatographies of DEAE-sephacel, sephacryl S-200 and hydroxyapatite. Although the 32 kda protein corresponded to [^<35>S]GTPgammaS binding activities was appeared in hydroxyapatite column chromatography, further purifications were necessary. In conclusion, there is the 32 kDa GTP binding protein in brain membranes as well as astrocytoma cells which is a candidate for a GTP binding pritein to activate phospholipase C in an IAP-insenitive manner. Less

本研究旨在阐明人星形细胞瘤细胞中激活磷脂酶C（PLase C）的GTP结合蛋白，该蛋白不是百日咳毒素（IAP）的底物。刺激TXA_2受体、毒蕈碱、H_1-组胺和缓激肽均能以IAP不敏感的方式激活PLase C。参与这些激动剂诱导的PLase C激活的GTP结合蛋白不是IAP的底物。用激动剂预处理细胞引起GTP γ S诱导的肌醇磷酸（IP）在膜制剂中的积累减少，反映了激动剂处理完整细胞后信号转导的功能减少。蔗糖梯度分离后膜的重峰部分含有受体和GTP结合蛋白。用激动剂处理完整细胞后，受体与GTP结合蛋白一起从重膜部分移动到轻峰部分。GTP结合蛋白在Heavy ...更多信息激动剂处理后的γ峰部分通过用[^ S] GTP γ S的光亲和标记进行分析<35>。用激动剂处理完整细胞后，32 kDa GTP结合蛋白在重峰部分中减少。32 kDa GTP结合蛋白可能参与受体介导的磷脂酶C激活。然后，从猪脑膜中纯化32 kDa GTP结合蛋白。在膜中存在32 kDa GTP结合蛋白，通过用[^ P] α-GTP的光亲和标记来确定<32>。32 kDa GTP结合蛋白用1% lubrol从胆酸盐浸提的膜中溶解。32 kDa的GTP结合蛋白可能是疏水性的，并且不被IAP ADP核糖基化。通过DEAE-Sephacel、Sephacryl S-200和羟基磷灰石柱层析纯化32 kDa GTP结合蛋白。虽然羟基磷灰石柱层析得到了32 kda的蛋白，其大小与[^<35>S] GTP γ S结合活性相当，但仍需进一步纯化。总之，在脑细胞膜以及星形细胞瘤细胞中存在32 kDa GTP结合蛋白，其是GTP结合蛋白以IAP不敏感方式激活磷脂酶C的候选者。少