Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit

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Abstract
The cyclic GMP physphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its .gamma. subunits and is activated following illumination by the GTP form of the .alpha. subunit of transducin (Ta-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme is .alpha..beta..gamma.2, Ta-GTP and .gamma. act reciprocally. We have investigated the activation mechanisms using fluorescein-labelled .gamma. subunit (.gamma.F) as a probe. .gamma.F 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). .gamma.F, like native .gamma., inhibited the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by .gamma.F was overcome by further addition of Ta-GTP. .gamma.F binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. .gamma.F added to ROS membrances became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that .gamma.F 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 .apprx. 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of .gamma. from the inhibitory sites. The binding of .gamma.F to trypsin-activated PDE.alpha..beta. was monitored by steady-state emission anisotropy measurements and compared with PDE activity. The results indicate that occupancy of each .gamma. binding site suppresses about half of the total activity of PDE.alpha..beta.; the dissociation constants for these sites are similar (.apprx. 10 pM). Our results fit a simple model in which T.alpha.-GTP interacts first with an .alpha..beta..gamma..gamma. holoenzyme and carries away one of the .gamma. subunits, to form a partially active PDE.alpha..beta..gamma. complex. This complex can then be converted to fully active PDE.alpha..beta. when a second .gamma. subunit is carried away by another T.alpha.-GTP. The T.alpha.-PDE.gamma. complex stays bound to the membrane.

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