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Properties of physiological roles of a GTP-binding protein which is associated physically and functionally with phospholipase C

与磷脂酶 C 物理和功能相关的 GTP 结合蛋白的生理作用特性

基本信息

批准号：
63571036
负责人：
TOYOSHIMA Satoshi
金额：
$ 1.34万
依托单位：
University of Tokyo
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-63571036/
关键词：
Phospholipase C GTP-binding protein Lymphocyte Mitogen ホスホリパーゼC マイトーゲン

项目摘要

Recently, we found that there is a novel GTP-binding protein (G-protein) in calf thymocytes which is associated not only physically but also functionally with soluble phosphoinositide-specific phospholipase C (PLC). It is quite possible that this G-protein is involved in the receptor-coupled regulation of PLC activity. In the present study, we purified this G-protein and investigated its properties. Furthermore, we also examined the physical and functional interactions between mitogen receptors(s), G-proteins and PLC.In addition to the ability to bind[^<35>S]GTPgammaS, the G-protein exhibited GTPase activity. The G-protein showed a molecular weight of 25,000 on gel filtration, which was different from those of well known G-proteins such as Gs, Gi, Go and Gt.Since we found that the G-protein might exist in the membrane fraction in a larger amount than in cytosol, this G-protein was purified from the calf thymocyte membrane. The purified G-protein (G21K) showed a molecular weight of 21,0 … More 00 on SDS-PAGE. Comparison of the partial amino acid sequences of G21K with those of known lower molecular weight G- proteins suggests that, although structurally similar to rho gene products, this is a novel G-protein.Then the mechanism underlying the receptor-coupled PLC activation was examined. We found that mitogen (concanavalin A) receptor(s) possibly interacts with at least two kinds of G-proteins (Gi and a low molecular weight G-protein). The low molecular weight G-protein might be the G-protein involved in receptor-coupled PLC activation and Gi might be responsible for the inhibitory regulation of the cellular cAMP level which inhibits PLC activity. Furthermore, we also found that a part of the G-protein with an approximate molecular weight of 25,000 might be translocated from the membrane to the cytosol and then enhance the cytosol PLC and/or membrane PLC exposed to the cytoplasmic side. At present, it is not evident whether G21K is the G-protein which is associated with mitogen receptor and translocates from membrane to cytosol by mitogen stimulation. However, it is quite possible because both G21K and the receptor associated G-protein were ADP-ribosylated with botulism toxin. Less

最近，我们发现在小牛胸腺细胞中存在一种新的GTP结合蛋白(G蛋白)，它不仅在物理上而且在功能上与可溶性磷脂酶C(PLC)相关。这种G蛋白很可能参与了PLC活性的受体偶联调节。在本研究中，我们纯化了这一G蛋白，并对其性质进行了研究。此外，我们还研究了丝裂原受体(S)、G蛋白和PLC之间的物理和功能相互作用。除了与GTP-Gamma结合的能力外，G-蛋白还具有GTP酶活性。凝胶过滤显示G蛋白的相对分子质量为25000，这与著名的G蛋白如Gs、GI、GO和GT不同。由于我们发现G蛋白可能以更多的形式存在于膜组分中，而不是胞浆中，因此从小牛胸腺细胞膜中纯化了该G蛋白。纯化的G蛋白(G21K)的相对分子质量为21，0…在SDS-PAGE上有00多个。将G21K蛋白的部分氨基酸序列与已知的低分子量G蛋白的部分氨基酸序列进行比较，发现G21K蛋白与Rho基因产物在结构上相似，但仍是一种新的G蛋白。我们发现有丝分裂原(刀豆蛋白A)受体(S)可能与至少两种G蛋白(Gi和一种低分子G蛋白)相互作用。低分子量G蛋白可能是参与受体偶联PLC激活的G蛋白，GI可能参与细胞内cAMP水平的抑制调节，从而抑制PLC活性。此外，我们还发现，分子量约为25000的G蛋白的一部分可能从细胞膜转移到胞浆，进而增强胞浆PLC和/或膜PLC暴露在细胞质一侧。目前，G21K是否是与丝裂原受体相关，通过丝裂原刺激从细胞膜转运到胞浆的G蛋白尚不清楚。然而，这是很有可能的，因为G21K和受体相关的G蛋白都是ADP-核糖化的肉毒杆菌毒素。较少