ACTIVATION MECHANISM OF RETINAL ROD CYCLIC-GMP PHOSPHODIESTERASE PROBED BY FLUORESCEIN-LABELED INHIBITORY SUBUNIT

The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its γ subunits and is activated following illumination by the GTP form of the α subunit of transducin (Tα-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme is αβγ2. Tα-GTP and γ act reciprocally. We have investigated the activation mechanism using fluorescein-labeled γ subunit (γF) as a probe. γF containing a single covalently attached fluorescein was prepared by reaction of PDE with 5-(iodoacetamido)fluorescein and purification by reversed-phase high-pressure liquid chromatography (HPLC). γF, like native y, inhibits the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by γF was overcome by further addition of Tα-GTP. y? binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. γF added to ROS membranes became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that γF became part of the PDE holoenzyme. Incorporation occurred in less than 1 min in the presence of light and GTP, but much more slowly (t1/2 ~ 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of γ from the inhibitory sites. The binding of γF to trypsin-activated PDEαβ was monitored by steady-state emission anisotropy measurements and compared with PDE activity. The results indicate that occupancy of each γ binding site suppresses about half of the total activity of PDEαβ; the dissociation constants for these sites are similar (~10 pM). Our results fit a simple model in which Tα-GTP interacts first with an αβγγ holoenzyme and carries away one of the γ subunits, to form a partially active PDEαβγ complex. This complex can then be converted to fully active PDEαβ when a second γ subunit is carried away by another Tα-GTP. The Tα-PDEγ complex stays bound to the membrane. © 1990, American Chemical Society. All rights reserved.