Purification, reconstitution on lipid vesicles, and assays of PDE6 and its activator G protein, transducin.
PDE6 in rod and cone photoreceptors is the principal effector of phototransduction. It is kept at a very low activity level in the dark, and in the light it is strongly activated by the guanosine 5'-triphosphate-bound form of the alpha-subunit of the G protein, transducin. Both transducin and PDE6 are peripheral proteins, and understanding both their interactions with one another and the roles of lipids in their function requires reconstituting purified proteins on the surfaces of defined lipid bilayers. We describe here methods for purifying the proteins, reconstituting them with vesicles, and assaying catalytic activity and binding.
Verna and Marrs McLean Department of Biology and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
Theodore G
TG
Wensel
Feng
F
He
Justine A
JA
Malinski
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D005136:Eye Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008567:Membranes, Artificial; D017949:Retinal Cone Photoreceptor Cells / Q000201:enzymology*; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D015293:Transducin / Q000737:chemistry*
D005136:Eye Proteins; D008567:Membranes, Artificial; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin