Abstract
Purpose::
Rod phosphodiesterase (PDE6), the effector enzyme amplifying visual signaling in rod and cone photoreceptors, is unique among the phosphodiesterase families not only for the heterodimeric (Pαß) structure of its catalytic subunits, but also for the two γ subunits (Pγ) whose inhibitory action can be relieved by binding to the light-activated G-protein, transducin. PDE6 is most closely related to PDE5 in structural, biochemical and pharmacological properties. This similarity allowed the use of the radiolabeled PDE5/6 inhibitors to test whether both catalytic sites are functional and if both sites are activated by transducin.
Methods::
Bovine rod outer segments (ROS) and purified PDE6 were prepared from frozen bovine retinas using standard procedures. Pαß was prepared by limited proteolysis of purified PDE6. Recombinant Pγ was expressed in bacteria and purified to >95% purity. The standard membrane filtration assay previously used for cGMP binding studies was adapted to permit quantitation of [3H]vardenafil (a kind gift of Bayer Healthcare AG) binding to PDE6.
Results::
[3H]vardenafil binds with high affinity (Kd < 10 nM) to the active sites of Pαß heterodimer. The maximum number of binding sites was found to be 1.9 ± 0.3 vardenafil per Pαß, showing that both Pα and Pß subunits can bind the drug. Addition of Pγ to Pαß blocks both the hydrolytic activity and [3H]vardenafil binding. Rod PDE6 associated with ROS membranes binds [3H]vardenafil only when transducin activates PDE6. The apparent affinity of vardenafil is lower for transducin-activated PDE6 than for Pαß. As PDE6 is progressively activated by transducin, [3H]vardenafil binding increases proportionally. However, [3H]vardenafil binding and hydrolytic activity never exceed 50% of the value for Pαß.
Conclusions::
Each catalytic domain in the Pαß heterodimer stoichiometrically binds radiolabeled inhibitor and possesses full catalytic activity. Transducin activated half of the catalytic sites on rod PDE6, suggesting that transducin relieves inhibition at only one catalytic site of PDE6.
Keywords: photoreceptors • enzymes/enzyme inhibitors • protein structure/function