Saito Natsumi, Ishida Yuko, Seno Keiji, Hayashi Fumio
Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan.
Protein Expr Purif. 2012 Mar;82(1):168-73. doi: 10.1016/j.pep.2011.12.007. Epub 2011 Dec 30.
cGMP phosphodiesterase 6 (PDE6) and rhodopsin kinase (GRK1) are quantitatively minor prenylated proteins involved in vertebrate phototransduction. Here, we report that methyl-β-cyclodextrin (MCD), a torus-shaped oligosaccharide with a hydrophobic pore, can be used as a selective extractant for such prenylated proteins from frog retinal disc membranes, and that MCD makes it possible to purify frog PDE6 holoenzyme with very simple procedure. The EC50s of MCD for the extraction of GRK1 and PDE6 from the cytoplasmic surface of the disc membrane were 0.17 and 5.1 mM, respectively. By successive extraction of the membrane by 1 mM and then 20 mM MCD, we obtained crude GRK1 and PDE6, respectively. From the 20mM extract, we were able to purify the PDE6 holoenzyme using one-step anion-exchange column chromatography. From 1mM MCD extract, GRK1 was further purified by an affinity column. Following the removal of MCD by ultrafiltration, we were able to confirm integrity of these enzymes by reconstituting phototransduction system in vitro. We have therefore demonstrated that MCD is a useful compound for selective extraction and purification of prenylated peripheral membrane proteins from the cytoplasmic surface of biological membranes.
环磷酸鸟苷磷酸二酯酶6（PDE6）和视紫红质激酶（GRK1）是脊椎动物光转导过程中含量较少的异戊二烯化蛋白。在此，我们报告称，甲基-β-环糊精（MCD），一种具有疏水孔的环状寡糖，可作为从蛙视网膜盘膜中选择性提取此类异戊二烯化蛋白的提取剂，并且MCD使得通过非常简单的程序纯化蛙PDE6全酶成为可能。MCD从盘膜细胞质表面提取GRK1和PDE6的半数有效浓度（EC50）分别为0.17 mM和5.1 mM。通过先后用1 mM和20 mM MCD提取膜，我们分别获得了粗制的GRK1和PDE6。从20 mM提取物中，我们能够通过一步阴离子交换柱色谱法纯化PDE6全酶。从1 mM MCD提取物中，GRK1通过亲和柱进一步纯化。通过超滤去除MCD后，我们能够通过体外重建光转导系统来确认这些酶的完整性。因此，我们证明了MCD是一种用于从生物膜细胞质表面选择性提取和纯化异戊二烯化外周膜蛋白的有用化合物。
