Fukada Y, Ohguro H, Saito T, Yoshizawa T, Akino T
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
J Biol Chem. 1989 Apr 5;264(10):5937-43.
During the process of transduction of a photon signal in vertebrate rod outer segments, transducin, a guanine nucleotide binding protein, mediates between a photobleaching intermediate of rhodopsin and a cGMP-phosphodiesterase. We report here that the beta gamma-subunit of bovine transducin (T beta gamma) characterized so far consists of two components (T beta gamma-1 and T beta gamma-2), which can be separated by anion exchange chromatography under nondenaturing conditions. Both components consisted of two polypeptides of Mr 36,000 (T beta) and about 8,000 (T gamma) in sodium dodecyl sulfate polyacrylamide (13%) gel electrophoresis. On a further analysis by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, T gamma subunits of T beta gamma-1 and T beta gamma-2 showed Mr values of 8,000 (T gamma-1) and 6,000 (T gamma-2), respectively. Amino acid compositions of both T gamma-1 and T gamma-2 roughly corresponded with that of T gamma previously reported and were quite different from that of gamma-subunit of cGMP-phosphodiesterase. Western blot analysis of freshly isolated rod outer segments by an antiserum raised against a mixture of T beta gamma-1 and T beta gamma-2 revealed the presence of both components in the membranes of a starting material. This observation excludes the possibility that one of the components might be produced artificially in the course of the purification. In the presence of a photobleaching intermediate of either unphosphorylated or phosphorylated rhodopsin, the binding of guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp) to the alpha-subunit of transducin (T alpha) was remarkably enhanced with increasing concentrations of purified T beta gamma-2. On the contrary, T beta gamma-1 retained little ability, if any, to enhance the GppNHp binding to T alpha; the ability of T beta gamma-1 was at least 30 times lower than that of T beta gamma-2. Such a low activity of T beta gamma-1 was attributed to inability for coupling of T alpha with a photobleaching intermediate of rhodopsin. These results indicate that T gamma-2 is essential for the GTP binding of transducin. The role of T gamma-1 in vertebrate photoreceptor cells was discussed.
在脊椎动物视杆细胞外段的光子信号转导过程中，转导素（一种鸟嘌呤核苷酸结合蛋白）在视紫红质的光漂白中间体与cGMP - 磷酸二酯酶之间起介导作用。我们在此报告，迄今为止所鉴定的牛转导素的βγ亚基（Tβγ）由两个组分（Tβγ - 1和Tβγ - 2）组成，在非变性条件下可通过阴离子交换色谱法分离。在十二烷基硫酸钠聚丙烯酰胺（13%）凝胶电泳中，两个组分均由Mr为36,000的两种多肽（Tβ）和约8,000的多肽（Tγ）组成。通过8M尿素/十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳进一步分析，Tβγ - 1和Tβγ - 2的Tγ亚基的Mr值分别为8,000（Tγ - 1）和6,000（Tγ - 2）。Tγ - 1和Tγ - 2的氨基酸组成大致与先前报道的Tγ的氨基酸组成相符，且与cGMP - 磷酸二酯酶的γ亚基的氨基酸组成有很大差异。用针对Tβγ - 1和Tβγ - 2混合物产生的抗血清对新鲜分离的视杆细胞外段进行蛋白质印迹分析，结果显示起始材料的膜中存在这两个组分。这一观察结果排除了其中一个组分可能在纯化过程中人为产生的可能性。在未磷酸化或磷酸化的视紫红质的光漂白中间体存在的情况下，随着纯化的Tβγ - 2浓度增加，鸟苷5'-（β，γ - 亚氨基）三磷酸（GppNHp）与转导素的α亚基（Tα）的结合显著增强。相反，Tβγ - 1增强GppNHp与Tα结合的能力（如果有的话）很小；Tβγ - 1的能力至少比Tβγ - 2低30倍。Tβγ - 1的这种低活性归因于Tα无法与视紫红质的光漂白中间体偶联。这些结果表明，Tγ - 2对于转导素的GTP结合至关重要。文中还讨论了Tγ - 1在脊椎动物光感受器细胞中的作用。
