Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-sepharose chromatography.
The biochemical bases for the differences in cone and rod photoreceptor physiology have not been thoroughly examined because of the difficulty in obtaining cone photoreceptor components. We report here the purification and preliminary characterization of a bovine cyclic GMP phosphodiesterase (PDE) which is enriched in cone photoreceptors. The cone PDE was purified at least 15,000-fold to apparent homogeneity from bovine retinas by DEAE-cellulose and cGMP-Sepharose affinity chromatography. The trypsin-activated cone PDE hydrolyzed cGMP with efficiency similar to that of the rod PDE. However, a number of characteristics distinguished the cone PDE from the rod isozyme including the subunit structure. As previously reported, the apparent molecular weight of the cone PDE large subunit (alpha') was slightly larger than either of the large subunits of the rod PDE (93,500 versus 88,000 and 84,000). Three other smaller polypeptides were associated with the alpha' subunit (Mr = 11,000, 13,000, and 15,000), one of which (11,000) may be identical to the rod PDE gamma subunit. Cone phosphodiesterase binds at least 10-fold more cyclic GMP/mol of PDE than the rod photoreceptor isozyme. Cyclic GMP binds to this noncatalytic site with high affinity (Kd = 11 nM) and dissociates very slowly (t1/2 = 10-20 min at 37 degrees C). Purified rod transducin activated the cone PDE in solution to at least 90% of the trypsin-activated level. The concentration of rod transducin required for half-maximal activation of cone PDE (15 nM) was 50-fold lower than that necessary for half-maximal activation of rod PDE. Thus several properties of the cone phosphodiesterase clearly distinguish it from the rod isozyme and could account for some differences in cone and rod physiology.
Gillespie
P G
PG
Department of Pharmacology, University of Washington, Seattle 98195.
Beavo
J A
JA
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000302:isolation & purification*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D006152:Cyclic GMP / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D010786:Photoreceptor Cells / Q000201:enzymology*; D015293:Transducin
D046911:Macromolecular Substances; D008565:Membrane Proteins; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin; D006152:Cyclic GMP