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

本研究旨在阐明在人星形细胞瘤细胞中激活磷脂酶C （PLase C）的GTP结合蛋白，而PLase C不是百日咳毒素（IAP）的底物。txa_2受体以及毒蕈碱、h_1组胺和缓激肽的刺激导致PLase C以IAP不敏感的方式激活。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可以减少gtpgammas诱导的膜制剂中肌醇磷酸（IP）的积累，这反映了完整细胞在激动剂处理后信号转导的功能减少。蔗糖梯度分离后的膜重峰部分含有受体和GTP结合蛋白。受体和GTP结合蛋白在用激动剂处理完整细胞后从重膜组分转移到轻峰组分。用[^<35>S]GTPgammaS光亲和标记分析了受体激动剂处理后GTP结合蛋白的减少。用激动剂处理完整细胞后，32 kDa的GTP结合蛋白在重峰分数中减少。32 kDa GTP结合蛋白可能参与了受体介导的磷脂酶c的激活。然后，从猪脑膜中纯化了32 kDa GTP结合蛋白。膜上有一个32 kDa的GTP结合蛋白，由[^<32>P] α -GTP光亲和标记确定。将32 kDa的GTP结合蛋白用1%的润滑油醇从提取的胆酸膜中溶解。32 kDa的gtp结合蛋白可能是疏水性的，并且不与IAP发生adp核糖基化。采用deae - sepacel、sepacryl S-200和羟基磷灰石柱层析纯化了32 kDa的GTP结合蛋白。虽然羟基磷灰石柱层析显示32 kda蛋白对应[^<35>S]GTPgammaS结合活性，但需要进一步纯化。综上所述，脑膜和星形细胞瘤细胞中存在32kda的GTP结合蛋白，该蛋白是GTP结合蛋白的候选蛋白，可以以不敏感的方式激活磷脂酶C。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