cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits.

Abstract

The cGMP phosphodiesterase (PDE) of cattie retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDEa and PDE.8, each around 85 kDa, and the light inhibitory one, PDEy or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the a subunit of transducin (Ta). The stoichiometric complex Ta-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDEa8-I2. (14) and preserved at -80'C. The thawed pellets were homogenized, illuminated, and then washed in medium salt buffer, to eliminate some minor proteins not relevant to this study. Extraction and "crude" purification were then carried out using the light, nucleotide, and ionic-strength dependence of binding of the species (14). Crude PDE was obtained by low ionic strength extraction of the illuminated membrane pellet and crude transducin was obtained by subsequent extraction of the same pellet after addition of 100 ,uM GTP[yS]. Total extract was obtained by direct low ionic strength extraction of an illuminated membrane pellet in the presence of GTP [yS]. Low salt buffer was 5 mM Hepes/1 mM dithiothreitol, pH 7.5; medium salt buffer was 20 mM Hepes/120 mM KCl/1 mM dithiothreitol, pH 7.5. Rhodopsin concentration in all extraction procedures was 2 mg/ml. GTP [yS] was used at 100 ,uM.Protein Chromatography. Separation and purification of transducin subunits, native PDE, and the various PDE units were performed as described (15), on an ion exchange column (Polyanion SI from Pharmacia) (unfortunately this very efficient column is not commercially available anymore, but we have a stock of them). Elution was obtained by Na2SO4 gradients (from 0 to 600 mM) in a buffer containing 20 mM Hepes, pH 7.5/1 mM MgSO4/5 mM 2-mercaptoethanol. Various gradient programs were used to optimize the separation of the different PDE species (see figure legends). Proteins were eluted from the column (0.5 ml/min) followed by UV absorption. Fractions (250 ,ul) 2424The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments

“…Although it is known that PDE activation terminates after the hydrolysis of T-bound GTP [1], numerous measurements carried out in reconstituted systems indicate that the rate of GTPase reaction is too slow (tens of seconds) (review [5,6]) compared with the real cascade quenching time in an ROS suspension (seconds) [7]. To overcome the above contradiction two possibilities were postulated: (i) the rate of T GTPase under physiological conditions is higher than in reconstituted systems [6,8,9]; (ii) the rapid PDE quenching in ROS is caused by a mechanism, independent of T GTPase [10,11].…”

Characteristics of Photoreceptor PDE (PDE6): similarities and differences to PDE5

“…4,5 Cone PDE6 differs from rod PDE6 in that its catalytic dimer is composed of two identical a 0 subunits. Also, the low molecular weight cone inhibitory g 0 subunits differ slightly in size (9.4 vs 9.7 kDa) and amino-acid composition from the rod g subunits.…”

Affinities of bovine photoreceptor cGMP phosphodiesterases for rod and cone inhibitory subunits

“…2, inset). At least two inhibitory subunits are believed necessary for full inhibition of rod PDE [5], so x-intercept values were divided by two to determine the concentration of active PDEy. The number of inhibitory subunits needed to fully inhibit the cone PDE has not been reported.…”

“…Tel-GTP dissociates from T/?y [4] and activates the PDE. Whether Ta-GTP actually binds and removes the PDE inhibitory subunits [5] or whether it binds and remains associated with the PDE [6,7] is still a matter of controversy. Within seconds of PDE activation, TOZ hydrolyzes GTP to GDP [8] and dissociates from the PDE, allowing PDEy to recombine with PDEaj?.…”

“…dimer is associated with at least two 11 kDa inhibitory (y) subunits [5]. This holoenzyme can be activated by trypsin which selectively degrades the PDEy subunits [l,lO].…”