Activated cGMP phosphodiesterase of retinal rods. A complex with transducin alpha subunit.

Purified G-protein (transducin) activated with the nonhydrolyzable analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and cGMP phosphodiesterase (PDE) from retinal rods are added to protein-stripped disc membranes. Specific binding of the mainly soluble alpha subunit of G-protein with GTP gamma S bound (G alpha GTP gamma S, activator of the PDE) to the disc membrane in the presence of PDE is measured from gel scans or experiments with labeled G-protein alpha subunit (G alpha). Its variation as a function of G concentration matches the theoretical variation of G alpha involved in the activation of PDE calculated with previously estimated dissociation constants (Bennett, N., and Clerc, A. (1989) Biochemistry 28, 7418-7424), and the G alpha bound/PDE ratio at saturation is close to 2. No increase of G alpha binding to the membrane is observed when purified inhibitory subunit of PDE (PDE gamma) is added together with or instead of total PDE, and excess PDE gamma remains soluble. These results suggest that activated PDE is a complex with the activator G alpha GTP rather than PDE from which the inhibitory subunits have been removed. A method for purifying PDE gamma with a high yield of recovery and activity is described.     

A monoclonal antibody to guanine nucleotide binding protein inhibits the light-activated cyclic GMP pathway in frog rod outer segments

A monoclonal antibody that blocks the light-activated cyclic GMP (cGMP) pathway in frog photoreceptor outer segments (ROS) has been obtained. The antibody (4A) inhibits guanine nucleotide binding to G-protein, the intermediate that links rhodopsin excitation to cGMP phosphodiesterase (PDE), inhibiting light-induced PDE activity as a consequence. Antibody inhibition of the light-activated cGMP pathway is complete at a stoichiometry of approximately one antibody per G-protein in the mixture, which indicates high specificity of the inhibition. Inhibition is more pronounced than that caused by PDE inhibitors such as isobutylmethylxanthine (IBMX) or Ro 20-1724. Antibody 4A has the further effect of inhibiting the phosphorylation of two low molecular weight proteins, components I and II, whose phosphorylation normally can be stimulated by raising cGMP levels. The inhibition is not overridden by adding cGMP, which suggests that the G-protein influences these phosphorylations by a pathway distinct from its action on cGMP concentration. Antibody 4A may prove useful as a probe of the relevance of the cGMP pathway to visual transduction in living photoreceptors. Six other monoclonal antibodies to G-protein, as well as six monoclonal antibodies to rhodopsin and one to PDE, do not block light-activated guanine nucleotide binding, PDE activity, or ROS protein phosphorylations.     

Effects of fluoride on retinal rod outer segment cGMP phosphodiesterase and G-protein

The effects of fluoride on ROS phosphodiesterase and G-protein have been studied using membrane-free extracts. When G-protein was present NaF, at millimolar concentrations, stimulated PDE activity however, in a G-protein free extract, cGMP hydrolysis was inhibited by high fluoride concentrations. Fluoride was also found to profoundly inhibit the ability of G-protein to bind a GTP analogue, GTP gamma S, both in the presence and absence of rhodopsin. Aluminium greatly modified these effects of fluoride on PDE and G-protein. The possibility that fluoride activates PDE through its effect on G-protein is discussed.     

Mechanism of transducin activation of frog rod photoreceptor phosphodiesterase. Allosteric interactiona between the inhibitory gamma subunit and the noncatalytic cGMP-binding sites

The rod photoreceptor phosphodiesterase (PDE) is unique among all known vertebrate PDE families for several reasons. It is a catalytic heterodimer (alphabeta); it is directly activated by a G-protein, transducin; and its active sites are regulated by inhibitory gamma subunits. Rod PDE binds cGMP at two noncatalytic sites on the alphabeta dimer, but their function is unclear. We show that transducin activation of frog rod PDE introduces functional heterogeneity to both the noncatalytic and catalytic sites. Upon PDE activation, one noncatalytic site is converted from a high affinity to low affinity state, whereas the second binding site undergoes modest decreases in binding. Addition of gamma to transducin-activated PDE can restore high affinity binding as well as reducing cGMP exchange kinetics at both sites. A strong correlation exists between cGMP binding and gamma binding to activated PDE; dissociation of bound cGMP accompanies gamma dissociation from PDE, whereas addition of either cGMP or gamma to alphabeta dimers can restore high affinity binding of the other molecule. At the active site, transducin can activate PDE to about one-half the turnover number for catalytic alphabeta dimers completely lacking bound gamma subunit. These results suggest a mechanism in which transducin interacts primarily with one PDE catalytic subunit, releasing its full catalytic activity as well as inducing rapid cGMP dissociation from one noncatalytic site. The state of occupancy of the noncatalytic sites on PDE determines whether gamma remains bound to activated PDE or dissociates from the holoenzyme, and may be relevant to light adaptation in photoreceptor cells.     

A functional link between the dark Mg-ATPase activity and the light-induced enzymatic cascade in rod outer segments

The characterization of a light-induced scattering change in suspensions of rod fragments, which requires previous swelling of the disks by the dark Mg-ATPase described by Uhl et al. [FEBS Lett. 107, 317-322 (1979)] is reported here. Reconstitution experiments demonstrate that this signal is dependent on the presence of G-protein, GTP and cGMP phosphodiesterase. Fast reversal associated with regenerability requires in addition the presence of some protein(s) of the cytoplasm (probably the rhodopsin kinase) and ATP. The amount of excited rhodopsin which saturates the signal is the same as that which saturates the previously described 'dissociation signal' [Kühn et al. (1981) Proc. Natl Acad. Sci. USA 78, 6873-6877] associated with the formation of the phosphodiesterase activator G alpha GTP (alpha subunit of the G-protein with GTP bound). The kinetics of the signal is slightly slower than that of the dissociation signal and its amplitude is proportional to the extent of swelling of the disks. These results suggest that the interaction between G alpha GTP and the phosphodiesterase modifies some structural feature of the disks and provide evidence for the existence of a functional link between the dark Mg-ATPase and the light-induced enzymatic cascade.     

Visual transduction in rod outer segments

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.     

Light-stimulated protein movement in rod photoreceptor cells of the rat retina

We examined the intracellular distribution of three proteins involved in the cyclic GMP cascade of visual transduction; cGMP phosphodiesterase, the alpha-subunit of G-protein and arrestin. In adult rats, light-induced changes in the amounts of G and arrestin in the photoreceptor cell outer segments were observed both by polyacrylamide gel analysis of purified ROS and by immunocytochemical localization on retinal sections. In dark conditions, G was concentrated in the outer segments of photoreceptor cells while in the light G alpha was seen in the inner segments and the outer nuclear layer. Arrestin had the opposite distribution, appearing in the inner segments and outer nuclear layer under dark conditions and in the ROS under light conditions. In contrast, PDE, the enzyme which is activated by G and inhibited by arrestin showed no light-stimulated movement. In both light- and dark-adapted retinas, PDE was localized primarily in the outer segments of the photoreceptor cells.     

Light-induced activation of the rod phosphodiesterase leads to a rapid transient increase of near-infrared light scattering

The so-called AT-signal described here is a transient light-induced increase of the near-infrared scattering from isolated bovine rod outer segments (ROS). Freshly prepared ROS are permeabilized with 0.01% Triton X-100 immediately before measurement in the presence of 1 mM GTP. The signal amplitude is saturated when approximately 2 rhodopsin molecules out of 30 000 are photo-excited. The signal recovers rapidly (approximately 90 s) and can be repeated in a succession of flashes. The AT-signal can be prevented by pre-activation of the phosphodiesterase (PDE) enzyme cascade at various levels: either at the level of G-protein, using ALF4- in darkness or GTP gamma S plus light; or at the level of the PDE catalytic unit, using protamine as an activator. The light sensitivity and kinetics of the AT-signal are similar to published parameters of PDE activation. These data suggest that light-induced activation of the PDE is the key reaction for the generation of the signal. On the other hand, blocking of the catalytic cGMP binding site by isobutylmethylxanthine only slightly affects the signal. We propose that the AT-signal reflects a structural change linked to the transient removal of the PDE inhibitory subunit from the catalytic unit.     

Light-induced conformational change in rhodopsin detected by modification of G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.     

Contribution of the guanosinetriphosphatase activity of G-protein to termination of light-activated guanosine cyclic 3',5'-phosphate hydrolysis in retinal rod outer segments

Light activation of GTP binding to G-protein and its eventual hydrolysis are hypothesized to lead to activation and inactivation of cGMP phosphodiesterase (PDE) in vertebrate rod disk membranes (RDM). However, the reported GTPase rate of 3 per minute is too slow to account for the observed rapid inactivation of PDE. Our investigations on GTPase activity showed that RDM isolated in the dark have considerable dark GTPase activity, which is enhanced by light. In dark and light, the enzyme exhibits biphasic substrate dependence with two Km's for GTP of 2-3 and 40-80 microM at 22 degrees C and less than 1 and 10-25 microM at 37 degrees C. The Km's were not influenced by light. On the basis of G-protein content of the RDM, the Vmax's for the two activities at 37 degrees C in light are 4-5 and 20-30 GTPs hydrolyzed per minute per G-protein. RDM washed free of soluble and peripheral proteins do not have measurable GTPase activity in the dark or light. Purified G-protein alone also did not turn over GTP, apparently because bleached rhodopsin is required for it to bind GTP. Reconstitution of washed membranes with purified G-protein restores both the low- and high-Km GTPase activities. Inactivation of G-protein as measured by PDE turnoff and dissociation signal recovery is found to be faster at higher than lower [GTP], consistent with the observation that the higher GTPase activity associated with the higher Km alos resides in the G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-Regulation of Intracellular cGMP and cAMP in Cultured Human Corpus Cavernosum Smooth Muscle Cells

The goal of this study was to assess the potential cross-regulation of cyclic nucleotides in human corpus cavernosum (HCC). Incubation of primary cultures of HCC smooth muscle cells with either the NO donor sodium nitroprusside (SNP, 10 μM) or the phosphodiesterase type 5 (PDE 5) inhibitor sildenafil (50 nM) produced little or no changes in the intracellular cGMP levels. Incubation with both SNP and sildenafil produced marked increases in cGMP. Interestingly, incubation of cells with 10 μM of forskolin or PGE₁ produced significant enhancement of cGMP accumulation. These increases were not further enhanced by the addition of SNP and sildenafil. Kinetic analyses of cGMP hydrolysis by PDE 5 showed that high concentrations of cAMP reversibly inhibited the enzyme with a K_i of 258 ± 54 μM. The increase in cGMP levels in response to cAMP generating agents is not due to assay artifact since cAMP did not cross-react with cGMP antibody. Our data suggest that cAMP up-regulates intracellular levels of cGMP, in part, by inhibition of PDE 5. We also noted that cGMP down-regulates cAMP synthesis via a mechanism requiring G-protein coupling of adenylyl cyclase. These observations may have important implications in the utility of pharmacotherapeutic agents targeting cyclic nucleotide metabolism for the treatment of erectile dysfunction.     

Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization

Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.     

cGMP influences guanine nucleotide binding to frog photoreceptor G-protein

A rapid light-induced decrease in cGMP is thought to play a role in regulating the permeability or light sensitivity of photoreceptor membranes. Photo-excited rhodopsin activates a guanine nucleotide-binding protein (G-protein) by catalyzing the exchange of bound GDP for GTP. This G-protein X GTP complex activates the phosphodiesterase resulting in a decrease in cGMP concentration. We have observed two processes in vitro which may be relevant for the regulation of G-protein activation. First, we have found that free GDP binds to G-protein with an affinity similar to that of GTP. These two nucleotides appear to compete for a common site. Since G-protein X GDP does not activate phosphodiesterase, light-induced changes in the GTP/GDP ratio known to occur on illumination may serve to reduce G-protein activation and hence reduce phosphodiesterase activation. Second, addition of cGMP in the presence of equimolar GTP and GDP causes GTP binding to G-protein to be enhanced compared to GDP binding. This effect increases as the cGMP concentration is increased from 0.05 to 2 mM. Thus, light-induced decreases in cGMP concentration may also act as a feedback control in reducing G-protein activation. One or both of these processes may be involved in the desensitization (light adaptation) of rod photoreceptors.     

Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin)

The mechanism of activation of cGMP phosphodiesterase by the GTP-binding protein in the disc membrane of retinal rods has been investigated by measuring the light-induced phosphodiesterase activity in reconstituted systems where the concentration of either the GTP-binding protein or the phosphodiesterase is varied. The results are consistent with the existence of two activator sites per phosphodiesterase functional unit: binding of one G alpha GTP (alpha subunit of the G-protein with GTP bound) with high affinity (100 +/- 50 nM) partially activates the enzyme (Vmax1 approxmately 0.05 Vmax to 0.10V max to trypsin-activated phosphodiesterase); binding of a second G alpha GTP with lower affinity (600 +/- 100 nM) induces maximal activation (Vmax2 approximately Vmax of trypsin-activated phosphodiesterase). The two different states of activated phosphodiesterase have the same Km for cGMP and the same pH dependence; they differ in their sensitivity to GMP. Micromolar concentration of protamines increases the affinity of the two activator sites and slightly increases Vmax1. When G-protein is activated with GTP-gamma S instead of GTP, the affinities of the two activator sites are not significantly modified, while Vmax1 appears to be increased.     

The effect of GDP on rod outer segment G-protein interactions

The role of GDP has heretofore been little studied in the analysis of visual receptor G-protein (G) interactions. Here we use kinetically resolved absorption and light scattering spectroscopy, centrifugation, porous membrane filtration, and enzyme assay to compare the effectiveness of GDP with that of GTP or gamma-thio-guanosine-5'-triphosphate in the modulation of G-protein binding to rod disc membranes and activated receptor (R*). We also compare effectiveness of GDP with that of GTP in the separation of G alpha and G beta gamma subunits and in activation of effector, cGMP phosphodiesterase. We find that when different nucleotide affinities are taken into account, actions such as the release of G from R* binding, earlier ascribed to GTP alone, are also typical of GDP. The principal specific actions of GTP that occur only weakly or undetectably for GDP are, respectively, the release of G-protein subunits from the membrane into solution and activation of phosphodiesterase. While GDP, like GTP, releases G-protein binding to receptor, we argue that GDP cannot mediate G-protein subunit separation, even on the membrane surface. GDP retained on G-protein after GTP hydrolysis may function to prevent tight binding to quiescent receptors in a manner analogous to its action on G-protein binding to activated receptors. Weak binding of G.GDP may function to accelerate receptor catalyzed amplification during transduction.     

Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system

Rhodopsin kinase was extracted into a buffer containing 200 mM KCl and no MgCl2. The activity of the enzyme was stabilized with the use of a mixture of protease inhibitors, aprotinin, benzamidine, leupeptin, and pepstatin. The extract consisted of three major proteins of molecular weight (Mr) 65,000, 56,000, and 37,000, of which the Mr 65,000 protein was identified with the kinase activity since preparations containing the other proteins had no kinase activity and the Mr 65,000 protein was phosphorylated when the extract was incubated with ATP. A reconstituted cGMP phosphodiesterase (PDE) system consisting of peripheral protein-depleted rod disk membranes (RDM), GTP binding protein (G-protein), and PDE was used to test the effectiveness of the rhodopsin kinase preparation in mediating the ATP-dependent quench of light activation of PDE. In the absence of kinase, light-activated PDE activity lasted several minutes. In its presence, ATP and to a lesser extent GTP quenched the activation about as rapidly as in rod disk membranes. The influence of kinase was unaffected by increasing G-protein or PDE content of the reconstituted system but was slowed down by brighter flashes, showing that quench was caused by the inactivation of bleached rhodopsin and not of PDE or G-protein.     

A dominant-negative Galpha mutant that traps a stable rhodopsin-Galpha-GTP-betagamma complex

Residues comprising the guanine nucleotide-binding sites of the α subunits of heterotrimeric (large) G-proteins (Gα subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both Gα subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric Gα subunit (designated αT*) that is mainly comprised of the α subunit of the retinal G-protein transducin and a limited region from the α subunit of Gi1. The αT*(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type αT*, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on αT*(S43N). The αT*(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type αT*. Thus, GTP-bound αT*(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. αT*(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type αT*. Activation of the PDE by αT*(S43N) was unaffected if either R* or β1γ1 alone was present, whereas it was inhibited when R* and the β1γ1 subunit were added together. Overall, our studies suggest that the S43N substitution on αT* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTP-bound Gα subunit, and the β1γ1 complex.     

Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse.

The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.     

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.     

Enhancement of phototransduction protein interactions by lipid surfaces

The G protein cascade of vision depends on two peripheral membrane proteins: the G protein, transducin (G(t)), and cGMP phosphodiesterase (PDE). Each has covalently attached lipids, and interacts with transduction components on the membrane surface. We have found that their surface interactions are critically dependent on the nature of the lipid. Membranes enhance their protein-protein interactions, especially if electrostatic attraction is introduced with positively charged lipids. These interactions are less enhanced on highly curved surfaces, but are most enhanced by unsaturated or bulky acyl chains. On positively charged membranes, G(t) assembles at a high enough density to form two-dimensional arrays with short-range crystalline order. Cationic membranes also support extremely efficient activation of PDE by the GTPgammaS (guanosine 5'-O-(thiotriphosphate)) form of Galpha(t) (Galpha(t)-GTPgammaS), minimizing functional heterogeneity of transducin and allowing activation with nanomolar Galpha(t)-GTPgammaS. Quantification of PDE activation and of the amount of Galpha(t)-GTPgammaS bound to PDE indicated that G(t) activates PDE maximally when bound in a 1:1 molar ratio. No cooperativity was observed, even at nanomolar concentrations. Thus, under these conditions, the one binding site for Galpha(t)-GTPgammaS on PDE that stimulates catalysis must be of higher affinity than one or more additional sites which are silent with respect to activation of PDE.