Methyl-β-cyclodextrin is a useful compound for extraction and purification of prenylated enzymes from the retinal disc membrane.
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.
Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan.
Natsumi
N
Saito
Yuko
Y
Ishida
Keiji
K
Seno
Fumio
F
Hayashi
D000818:Animals; D002852:Chromatography, Ion Exchange; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6 / Q000302:isolation & purification* / Q000378:metabolism; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000302:isolation & purification* / Q000378:metabolism; D054876:Prenylation; D011892:Rana catesbeiana; D012160:Retina / Q000201:enzymology*; D047392:beta-Cyclodextrins / Q000737:chemistry*
D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D051606:G-Protein-Coupled Receptor Kinase 1; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6