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

本研究旨在阐明人脑星形细胞瘤细胞中激活磷脂酶C(Plase C)的GTP结合蛋白，该酶不是百日咳毒素(IAP)的底物。刺激TXA2受体、毒扁豆碱、H_1组胺和缓激肽可使IAP不敏感地激活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。用激动剂处理细胞可减少GTP-GammaS诱导的肌醇磷酸(IP)在膜制剂中的积聚，这反映在激动剂处理完整细胞后信号转导功能的降低。蔗糖梯度分离后的膜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，受体和GTP结合蛋白从重膜部分移至轻峰部分。HEAV…中谷氨酸蛋白结合蛋白的降低用[^&lt；35&gt；S]GTP-GammaS光亲和标记分析激动剂治疗后更多的γ峰分数。用激动剂处理完整细胞后，32 kDa GTP结合蛋白在重峰部分被降低。32 kDa GTP结合蛋白可能参与了受体介导的磷脂酶C激活。膜上有一个32 kDa的GTP结合蛋白，用[^&lt；32&gt；P]α-GTP进行光亲和标记。从胆酸抽提膜中提取的32 kDa GTP结合蛋白用1%的叶黄素溶解。32 kDa GTP结合蛋白可能是疏水性的，不与IAP发生ADP核糖化反应。经DEAESephacel、Sephacryl S-200和羟基磷灰石柱层析纯化得到32 kDa的GTP结合蛋白。尽管在羟基磷灰石柱层析中出现了与[^&lt；35&gt；S]GTP-GammaS结合活性相对应的32kda蛋白，但仍需进一步纯化。综上所述，在脑膜和星形细胞瘤细胞中存在32 kDa的GTP结合蛋白，这是一种候选的GTP结合蛋白以IAP非敏感的方式激活磷脂酶C。较少

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