Department of Ophthalmology, Kresge Eye Institute, Wayne State University, 4717 St. Antoine St., Detroit, MI 48201, USA.
Mol Cell Biochem. 2010 Jun;339(1-2):235-51. doi: 10.1007/s11010-010-0404-y. Epub 2010 Feb 23.
Rod photoreceptor cGMP phosphodiesterase (PDE6) consists of a catalytic subunit complex (Palphabeta) and two inhibitory subunits (Pgamma). In the accompanying article, using bovine photoreceptor outer segment homogenates, we show that Pgamma as a complex with the GTP-bound transducin alpha subunit (GTP-Talpha) dissociates from Palphabetagammagamma on membranes, and the Palphabetagammagamma becomes Pgamma-depleted. Here, we identify and characterize the Pgamma-depleted PDE. After incubation with or without guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), Palphabeta complexes are extracted. When a hypotonic buffer is used, Palphabetagammagamma, Palphabetagamma, and a negligible amount of a Palphabeta complex containing Pgamma are isolated with GTPgammaS, and only Palphabetagammagamma is obtained without GTPgammaS. When an isotonic buffer containing Pdelta, a prenyl-binding protein, is used, Palphabetagammagammadelta, Palphabetagammadeltadelta, and a negligible amount of a Palphabeta complex containing Pgamma and Pdelta are isolated with GTPgammaS, and Palphabetagammagammadelta is obtained without GTPgammaS. Neither Palphabeta nor Palphabetagammagamma complexed with GTPgammaS-Talpha is found under any condition we examined. Palphabetagamma has approximately 12 times higher PDE activity and approximately 30 times higher Pgamma sensitivity than those of Palphabetagammagamma. These results indicate that the Pgamma-depleted PDE is Palphabetagamma. Isolation of Palphabetagammagammadelta and Palphabetagammadeltadelta suggests that one C-terminus of Palphabeta is involved in the Palphabetagammagamma interaction with membranes, and that Pgamma dissociation opens another C-terminus for Pdelta binding, which may lead to the expression of high PDE activity. Cone PDE behaves similarly to rod PDE in the anion exchange column chromatography. We conclude that the mechanisms for PDE activation are similar in mammalian and amphibian photoreceptors as well as in rods and cones.
视杆细胞 cGMP 磷酸二酯酶（PDE6）由一个催化亚基复合物（Palphabeta）和两个抑制亚基（Pgamma）组成。在随附的文章中，我们使用牛视杆细胞外段匀浆，表明 Pgamma 与结合 GTP 的转导蛋白 alpha 亚基（GTP-Talpha）形成复合物从膜上与 Palphabetagammagamma 分离，并且 Palphabetagammagamma 被耗尽 Pgamma。在这里，我们鉴定并表征了耗尽 Pgamma 的 PDE。在用或不用鸟苷 5'-O-(3-硫代三磷酸)（GTPgammaS）孵育后，提取 Palphabeta 复合物。当使用低渗缓冲液时，用 GTPgammaS 分离出含有 Pgamma 的 Palphabetagammagamma、Palphabetagamma 和少量 Palphabeta 复合物，而不用 GTPgammaS 则仅获得 Palphabetagammagamma。当使用含有 Pdelta 的等渗缓冲液（一种 prenyl-binding 蛋白）时，用 GTPgammaS 分离出含有 Pgamma 和 Pdelta 的 Palphabetagammagammadelta、Palphabetagammadeltadelta 和少量 Palphabeta 复合物，而不用 GTPgammaS 则仅获得 Palphabetagammagammadelta。在我们检查的任何条件下，都未发现与 GTPgammaS-Talpha 复合的 Palphabeta 或 Palphabetagammagamma 复合物。Palphabetagamma 的 PDE 活性比 Palphabetagammagamma 高约 12 倍，对 Pgamma 的敏感性高约 30 倍。这些结果表明，耗尽 Pgamma 的 PDE 是 Palphabetagamma。Palphabetagammagammadelta 和 Palphabetagammadeltadelta 的分离表明 Palphabeta 的一个 C 末端参与了 Palphabetagammagamma 与膜的相互作用，并且 Pgamma 的解离为 Pdelta 的结合打开了另一个 C 末端，这可能导致高 PDE 活性的表达。锥体 PDE 在阴离子交换柱层析中表现类似于杆 PDE。我们得出结论，PDE 激活的机制在哺乳动物和两栖动物光感受器以及棒状和锥状细胞中是相似的。
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