Ohoka Y, Imai S, Kozasa T, Maehama T, Takahashi K, Kaziro Y, Ui M, Katada T
Department of Life Science, Tokyo Institute of Technology, Kanagawa.
J Biochem. 1991 Mar;109(3):428-35. doi: 10.1093/oxfordjournals.jbchem.a123398.
A GTP-binding protein with an Mr of 24,000 was purified from a cholate extract of bovine brain membranes in addition to the previously reported alpha beta gamma-trimeric GTP-binding proteins (G proteins). Partial amino acid sequence analysis of the purified 24-kDa protein revealed that it was not identical to any of the low Mr GTP-binding proteins already reported, but similar to the rac-gene products serving as the substrate of an ADP-ribosyltransferase (C3) purified from the culture medium of Clostridium botulinum type C. However, the 24-kDa protein was not ADP-ribosylated by the botulinum C3 enzyme. The 24-kDa protein was purified as a nucleotide-free form and characterized by the following unique properties distinct from those of alpha beta gamma-trimeric G proteins. (1) Mg2+ was essentially required for nucleotide binding to the 24-kDa protein; there was a progressive increase in its binding affinity for nucleotides as the concentration of the divalent cation was increased. (2) Nucleotides previously bound to the 24-kDa protein were rapidly dissociated from the protein in Mg(2+)-free medium, in accord with the fact that the protein was indeed purified as a nucleotide-free form with Mg(2+)-free solutions. (3) The 24-kDa protein apparently exhibited much lower GTPase activity than do alpha beta gamma-trimeric G proteins because the product GDP was released from the 24-kDa protein in exchange for the substrate GTP only at a very low rate. Based on these findings, a possible role of the 24-kDa protein in cellular signalling is discussed in comparison with well characterized alpha beta gamma-trimeric G proteins.
除了先前报道的αβγ三聚体GTP结合蛋白（G蛋白）外，还从牛脑膜的胆酸盐提取物中纯化出一种分子量为24,000的GTP结合蛋白。对纯化的24 kDa蛋白进行的部分氨基酸序列分析表明，它与任何已报道的低分子量GTP结合蛋白都不相同，但与肉毒杆菌C型培养基中纯化的作为ADP核糖基转移酶（C3）底物的rac基因产物相似。然而，24 kDa蛋白不会被肉毒杆菌C3酶进行ADP核糖基化。24 kDa蛋白以无核苷酸形式纯化，并具有以下不同于αβγ三聚体G蛋白的独特性质。（1）Mg2+是核苷酸与24 kDa蛋白结合所必需的；随着二价阳离子浓度的增加，其与核苷酸的结合亲和力逐渐增加。（2）先前与24 kDa蛋白结合的核苷酸在无Mg(2+)的培养基中会迅速从该蛋白上解离，这与该蛋白确实是用无Mg(2+)溶液纯化得到的无核苷酸形式这一事实相符。（3）24 kDa蛋白的GTPase活性明显低于αβγ三聚体G蛋白，因为只有在非常低的速率下，产物GDP才会从24 kDa蛋白上释放出来以交换底物GTP。基于这些发现，将24 kDa蛋白与特征明确的αβγ三聚体G蛋白进行比较，讨论了其在细胞信号传导中的可能作用。
