Activated cGMP phosphodiesterase of retinal rods. A complex with transducin alpha subunit.
Purified G-protein (transducin) activated with the nonhydrolyzable analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and cGMP phosphodiesterase (PDE) from retinal rods are added to protein-stripped disc membranes. Specific binding of the mainly soluble alpha subunit of G-protein with GTP gamma S bound (G alpha GTP gamma S, activator of the PDE) to the disc membrane in the presence of PDE is measured from gel scans or experiments with labeled G-protein alpha subunit (G alpha). Its variation as a function of G concentration matches the theoretical variation of G alpha involved in the activation of PDE calculated with previously estimated dissociation constants (Bennett, N., and Clerc, A. (1989) Biochemistry 28, 7418-7424), and the G alpha bound/PDE ratio at saturation is close to 2. No increase of G alpha binding to the membrane is observed when purified inhibitory subunit of PDE (PDE gamma) is added together with or instead of total PDE, and excess PDE gamma remains soluble. These results suggest that activated PDE is a complex with the activator G alpha GTP rather than PDE from which the inhibitory subunits have been removed. A method for purifying PDE gamma with a high yield of recovery and activity is described.
Clerc
A
A
Unité Associée 520 du Centre National de la Recherche Scientifique, Centre d'Etudes Nucléaires de Grenoble, France.
Bennett
N
N
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D015293:Transducin / Q000378:metabolism*
D008565:Membrane Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin