Purification of PDE6 isozymes from mammalian retina.
The photoreceptor phosphodiesterase (PDE6) is the central effector of visual transduction in vertebrate retinal photoreceptors. Distinct isozymes of PDE6 exist in rods and cones. Mammalian retina serves as an abundant source of tissue for PDE6 purification. Methods are described for the isolation and purification of membrane-associated PDE6 from rod outer segment membranes. Purification of cone PDE6 from the soluble fraction of retinal extracts is also described. Several procedures that can purify the rod and cone isozymes to homogeneity, including anion exchange, hydrophobic interaction, gel filtration, hydroxyapatite, and immunoaffinity chromatography, are presented. A method to activate PDE6 by limited proteolysis of its inhibitory gamma-subunit is also provided.
Pentia
Dana C
DC
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, USA.
Hosier
Suzanne
S
Collupy
Rachel A
RA
Valeriani
Beverly A
BA
Cote
Rick H
RH
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry* / Q000302:isolation & purification*; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry*; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry*
D007527:Isoenzymes; D015106:3',5'-Cyclic-GMP Phosphodiesterases