Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States.
Biochemistry. 2011 Nov 8;50(44):9520-31. doi: 10.1021/bi2014695. Epub 2011 Oct 14.
Membrane-bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the α and β subunit catalytic core, interactions of the core with two intrinsically disordered, proteolysis-prone inhibitory PDEγ (Pγ) subunits, and binding of two types of isoprenyl-binding protein δ, called PrBP/δ, to the isoprenylated C-termini of the catalytic core. Structural studies of native PDE6 have been also been hampered by the lack of a heterologous expression system for the holoenzyme. In this work, we purified PDE6 in the presence of PrBP/δ and screened for additives and detergents that selectively suppress PDE6 basal activity while sparing that of the trypsin-activated enzyme. Some detergents removed PrBP/δ from the PDE complex, separating it from the holoenzyme after PDE6 purification. Additionally, selected detergents also significantly reduced the level of dissociation of PDE6 subunits, increasing their homogeneity and stabilizing the holoenzyme by substituting for its native membrane environment.
膜结合型磷酸二酯酶 6（PDE6）在调节视杆细胞中 cGMP 水平方面在视觉信号转导中起着重要作用。我们对 PDE6 催化作用和结构的理解受到对 α 和 β 亚基催化核心、核心与两个固有无序、易发生蛋白水解的抑制性 PDEγ（Pγ）亚基的相互作用以及两种类型的异戊烯基结合蛋白 δ（称为 PrBP/δ）与催化核心异戊烯化 C 末端的结合的不足描述。天然 PDE6 的结构研究也受到缺乏全酶异源表达系统的阻碍。在这项工作中，我们在存在 PrBP/δ 的情况下纯化了 PDE6，并筛选了选择性抑制 PDE6 基础活性而不抑制胰蛋白酶激活酶活性的添加剂和去污剂。一些去污剂将 PrBP/δ 从 PDE 复合物中去除，在 PDE6 纯化后将其与全酶分离。此外，选定的去污剂还显著降低了 PDE6 亚基的解离水平，通过取代其天然膜环境增加了它们的同质性并稳定了全酶。
