On a soluble system for studying light activation of rod outer segment cyclic GMP phosphodiesterase

Bovine rod outer segment (ROS) cyclic GMP phosphodiesterase (PDE) could be activated about 6-fold by light, an effect that could be simulated by isolated bleached rhodopsin. About 90% of PDE activity in ROS could be extracted with 10 mM Tris-HCl, pH 7.5, but light is ineffective in activating the soluble enzyme. However, bleached rhodopsin could activate it in the presence of a very low concentration of ATP, strongly suggesting the mediation of rhodopsin in the light activation of the enzyme in ROS. Direct evidence is presented to suggest that the phosphorylation of opsin (bleached rhodopsin) is unrelated to the activation of PDE by bleached rhodopsin and ATP. The reconstitution of the light activation of PDE in a soluble system presented here opens up a new direction to future investigations on the mechanism of light regulation of cyclic GMP levels in retina and its implication in the photoreceptor function.     

Mechanism of ATP quench of phosphodiesterase activation in rod disc membranes

GTP-dependent light activation of cyclic GMP phosphodiesterase in bovine rod disc membranes was quenched by ATP. ATP reduced both initial velocity (V0) and turn off time (toff) of phosphodiesterase activated by a flash that bleached 1.5 X 10(-5) of the rhodopsin present. In the absence of rhodopsin kinase, ATP had no effect on either V0 or toff of reconstituted preparations containing phosphodiesterase and GTP*-binding protein. Addition of partially purified rhodopsin kinase to such reconstitutions again permitted ATP to quench both initial velocity and turn off time. It is thus likely that kinase-mediated phosphorylation of bleached rhodopsin reduces and arrests light-induced phosphodiesterase activation. Thermolysin cleavage of rhodopsin's COOH-terminal dodecapeptide eliminated ATP's effect on toff, but did not diminish its effect on V0. Thus, the effects of ATP and kinase on V0 may be mediated by sites proximal to and effects on toff by sites distal to the thermolysin cleavage point at rhodopsin's COOH-terminal end.     

Stimulation of rhodopsin phosphorylation by guanine nucleotides in rod outer segments

Porcine rod outer segment (ROS) proteins were phosphorylated in the presence of [gamma-32P]ATP and Mg2+, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by autoradiography. The phosphorylation of rhodopsin, the major protein-staining band (Mr approximately 34 000-38 000), was markedly and specifically increased by exposure of rod outer segments to light; various guanine nucleotides (10 microM) including GMP, GDP, and GTP also specifically increased rhodopsin phosphorylation (up to 5-fold). Adenine nucleotides (cyclic AMP, AMP, and ADP at 10 microM) and 8-bromo-GMP (10 microM) or cyclic 8-bromo-GMP (10 microM) had no detectable stimulatory effect on rhodopsin phosphorylation. GTP increased the phosphorylation of rhodopsin at concentrations as low as 100 nM, and guanosine 5'-(beta, gamma-imidotriphosphate), a relatively stable analogue of GTP, was nearly as effective as GTP. Maximal stimulation of rhodopsin phosphorylation by GTP was observed at 2 microM. GMP and GDP were less potent than GTP. Both cyclic GMP and GMP were converted to GTP during the time period of the protein phosphorylation reaction, suggestive of a GTP-specific effect. Transphosphorylation of guanine nucleotides by [32P]ATP and subsequent utilization of [32P]GTP as a more effective substrate were ruled out as an explanation for the guanine nucleotide stimulation. With increasing concentrations of ROS proteins, the phosphorylation of rhodopsin was nonlinear, whereas in the presence of GTP (2 microM) linear increases in rhodopsin phosphorylation as a function of added ROS protein were observed. These results suggest that GTP stimulates the phosphorylation of rhodopsin by ATP and that a GTP-sensitive inhibitor (or regulator) of rhodopsin phosphorylation may be present in ROS.     

Interactions of nucleotide analogues with rod outer segment guanylate cyclase.

Light activation of cyclic GMP hydrolysis in rod outer segments is mediated by a G-protein which is active in the GTP-bound form. Substitution of GTP with a nonhydrolyzable GTP analogue is thought to leave the G-protein in a persistently activated state, thereby prolonging the hydrolysis of cyclic GMP. Restoration of cyclic GMP concentration in the cell also depends upon GTP since it is the substrate for guanylate cyclase, but little is known about the effects of GTP analogues on this enzyme. We report here the effects of the analogues of GTP and ATP as inhibitors and substrates of rod disk membrane guanylate cyclase. The rate of cyclic GMP synthesis from GTP in rod disk membranes was about 50 pmol min-1 (nmol of rhodopsin)-1. Analogues of GTP and adenine nucleotides competitively inhibited the cyclase activity. The order of inhibition, with magnesium as metal cofactor, was ATP greater than GMP-PNP greater than AMP-PNP approximately GTP-gamma-S; with manganese, AMP-PNP was more inhibitory than GTP-gamma-S. The inhibition constants, with magnesium as cofactor, were 0.65-2.0 mM for GTP-gamma-S, 0.4-0.8 mM for GMP-PNP, 1.5-2.3 mM for AMP-PNP, and 0.07-0.2 mM for ATP. The fraction of cyclase activity inhibited by analogues was similar at 1 and 0.03 microM calcium. Besides inhibition of cyclase, the analogues also served as its substrates. GTP-gamma-S substituted GTP with about 85% efficiency while GMP-PNP and ATP were about 5 and 7% as efficient, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of rhodopsin and quenching of cyclic GMP phosphodiesterase activation by ATP at weak bleaches

Light and GTP-dependent cyclic GMP phosphodiesterase activation of rod disk membranes is rapidly quenched by ATP. Maximum speed of this effect occurs only with the weakest bleaches. Though it has been proposed that ATP mediates its effect through rapid phosphorylation of bleached rhodopsin, previous workers have found phosphorylation kinetics too slow by more than an order of magnitude to be causal in quenching of cyclic GMP phosphodiesterase activation. In this report, we use preparations retaining more endogenous rhodopsin kinase, higher specific activity ATP, and cyclic GMP phosphodiesterase quenching conditions to show that ATP-dependent multiple phosphorylation of rhodopsin at very weak bleaches (10(-5)) is complete in less than 2 s, easily compatible with cyclic GMP phosphodiesterase quench times of 4 s measured under identical conditions. Thus, it seems likely that previous efforts to achieve high 32P counts by using large bleaches have produced conditions of substrate saturation where much longer times to completion are caused by a very large ratio of substrate to enzyme velocity. Such conditions are not appropriately compared to those that support rapid quenching. We conclude that the speed of rhodopsin phosphorylation is, in fact, adequate to explain ATP quenching of cyclic GMP phosphodiesterase activation.     

Purification and properties of guanylate kinase from bovine retinas and rod outer segments

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside-diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3). The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP in equilibrium GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20-23 kDa for both native enzyme and dodecylsulfate-denatured polypeptide), Km values (13 microM for GMP and 430 microM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s-1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.     

Influence of calcium on guanosine 3'',5''-cyclic monophosphate levels in frog rod outer segments

In the presence of 10(-9) M calcium, rod outer segments freshly detached from dark-adapted frog retinas contain between 0.01 and 0.02 moles of guanosine 3',5'-cyclic monophosphate (cyclic GMP) per mole of rhodopsin. The dark level of cyclic GMP is reduced approximately 50% by illumination that bleaches 5 x 10(5) rhodopsin molecules/outer segments. The dark levels of cyclic GMP also can be suppressed to approximately 0.007 mol/mol of rhodopsin by increasing the concentration of calcium from 10(-9) M to 2 x 10(-9) M, and they remain at this level as calcium concentration is raised to 10(-3) M. The final level to which illumination reduces cyclic GMP in unaffected by the calcium concentration between 10(-9) and 10(-3) M. The maximal light-induced decrease in cyclic GMP occurs within 1 s from the onset of illumination at all calcium concentrations. The magnitude and time-course of the light-induced decrease in cyclic GMP measured in these experiments are comparable to values obtained previously (Woodruff et al. 1977. J. Gen. Physiol. 69:677-679; Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). The data are consistent with a role for cyclic GMP in visual transduction irrespective of the calcium concentration.     

Kinetics of the Hydrolysis of 8-Bromo-Cyclic GMP by the Light-Activated Phosphodiesterase of Toad Rods

The hypothesis that cyclic GMP is the internal transmitter of retinal rod phototransduction, when combined with the observations that 8-bromo-cyclic GMP opens the cyclic GMP-dependent outer segment conductance and that rods into which 8-bromo-cyclic GMP has been injected still respond to light, predicts that the light-activated phosphodiesterase (EC 3.1.4.17) must catalyze the hydrolysis of 8-bromo-cyclic GMP. This hypothesis was tested by measuring light-activated toad rod disk membrane phosphodiesterase with a pH assay technique. Phosphodiesterase-catalyzed hydrolysis of 8-bromo-cyclic GMP was confirmed: at pH 8.0, total proton production after flash activation was identical to total amount of 8-bromo-cyclic GMP added as substrate. Photoactivated phosphodiesterase was remarkably less efficient in catalyzing the hydrolysis of 8-bromo-cyclic GMP than of cyclic GMP: Vmax for 8-bromo-cyclic GMP was 0.063 M/M rhodopsin/s, whereas that for cyclic GMP was 11 M/M rhodopsin/s--170 times greater. The Km for 8-bromo-cyclic GMP was 160 microM, and for cyclic GMP, 590 microM. 8-bromo-cyclic GMP competitively inhibited phosphodiesterase-catalyzed hydrolysis of cyclic GMP with a Ki of 1.2 mM. Complete reaction progress curves were analyzed for obedience to Michaelis-Menten kinetics: cyclic GMP hydrolysis, 8-bromo-cyclic GMP hydrolysis, and cyclic GMP hydrolysis in the presence of 8-bromo-cyclic GMP as competitive inhibitor were found to follow the integrated form of the Michaelis-Menten equation over the time course of the reactions, assuming phosphodiesterase was activated as a step. The kinetic parameters extracted from reaction progress curves were consistent with those derived from analysis of the initial velocity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein complement of rod outer segments of frog retina

Rod outer segments (ROS) from frog retina have been purified by Percoll density gradient centrifugation, a procedure that preserves their form and intactness. One- and two-dimensional electrophoretic analysis reveals a smaller number of proteins than is observed in many cell organelles and permits quantitation of the 20 most abundant polypeptides. Rhodopsin accounts for 70% of the total protein (3 X 10(9) copies/outer segment), and approximately 70 other polypeptides are present at more than 6 X 10(4) copies/outer segment. Another 17% of the total protein is accounted for by the G-protein (3 X 10(8) copies/outer segment) that links rhodopsin bleaching and the activation of cyclic GMP phosphodiesterase (PDE). The phosphodiesterase accounts for 1.5% of the protein (1.5 X 10(7) copies/outer segment), and a 48,000-dalton component that binds to the membrane in the light accounts for a further 2.6%. The function of approximately 90% of the total protein in the outer segment is known, and two-thirds of the non-rhodopsin protein is accounted for by enzyme activities associated with cyclic GMP metabolism. The relative molar abundance of rhodopsin, G-protein, and PDE is 100:10:1. Apart from these major membrane-associated proteins, most of the other proteins are cytosolic. Thirteen other polypeptides are found at an abundance of one or more copies per 1000 rhodopsins, nine soluble and four membrane-bound, and their abundance relative to rhodopsin has been quantitated. ROS have been separated into subcellular fractions which resolve three classes of soluble, extrinsic membrane, and integral membrane proteins. A listing of the proteins that are phosphorylated and their subcellular localization is given. Approximately 25 phosphopeptides are detected, and most are in the soluble fraction. Fewer phosphorylated proteins are associated with the purified outer segments than with crude ROS. Distinct patterns of phosphorylation are associated with intact rods incubated with [32P]Pi and broken rods incubated with [gamma-32P]ATP.     

Guanosine 3'',5''-cyclic monophosphate and the in vitro physiology of frog photoreceptor membranes

Frog rod outer segments freshly detached from dark-adapted retinas contain approximately 1-2 molecules of guanosine 3',5'-cyclic monophosphate (cyclic GMP) for every 100 molecules of visual pigment present. This cyclic GMP decays to 5'-GMP, and the conversion is accelerated upon illumination of the outer segments. Bleaching one rhodopsin molecule can lead to the hydrolysis of 1,000-2,000 molecules of cyclic GMP within 100-300 ms. The decline in cyclic GMP concentration becomes larger as illumination increases, and varies with the logarithm of light intensity at levels which bleach between 5 X 10(2) and 5 X 10(5) rhodopsin molecules per outer segment-second. Light suppression of plasma membrane permeability, assayed in vitro as light suppression of outer segment swelling in a modified Ringer's solution, occurs over this same range of light intensity. The correlation between cyclic GMP and permeability or swelling is maintained in the presence of two pharmacological perturbations: papaverine, a phosphodiesterase inhibitor, increases both cyclic GMP levels and the dark permeability of the plasma membrane; and beta,gamma-methylene ATP increases the effectiveness of light in suppressing both permeability and cyclic GMP levels.     

Calcium and cyclic GMP regulation of light-sensitive protein phosphorylation in frog photoreceptor membranes

In frog photoreceptor membranes, light induces a dephosphorylation of two small proteins and a phosphorylation of rhodopsin. The level of phosphorylation of the two small proteins is influenced by cyclic GMP. Measurement of their phosphorylation as a function of cyclic GMP concentration shows fivefold stimulation as cyclic GMP is increased from 10(-5) to 10(-3) M. This includes the concentration range over which light activation of a cyclic GMP phosphodiesterase causes cyclic GMP levels to fall in vivo. Cyclic AMP does not affect the phosphorylations. Calcium ions inhibit the phosphorylation reactions. Calcium inhibits the cyclic GMP-stimulated phosphorylation of the small proteins as its concentration is increased from 10(-6) to 10(-3) M, with maximal inhibition of 70% being observed. Rhodopsin phosphorylation is not stimulated by cyclic nucleotides, but is inhibited by calcium, with 50% inhibition being observed as the Ca++ concentration is increased from 10(-9) to 10(-3) M. A nucleotide binding site appears to regulate rhodopsin phosphorylation. Several properties of the rhodopsin phosphorylation suggest that it does not play a role in a rapid ATP-dependent regulation of the cyclic GMP pathway. Calcium inhibition of protein phosphorylation is a distinctive feature of this system, and it is suggested that Ca++ regulation of protein phosphorylation plays a role in the visual adaptation process. Furthermore, the data provide support for the idea that calcium and cyclic GMP pathways interact in regulating the light-sensitive conductance.     

Light adaption of the cyclic GMP phosphodiesterase of frog photoreceptor membranes mediated by ATP and calcium ions

The light-activated guanosine 3',5'-cyclic monophosphate (cyclic GMP) phosphodiesterase (PDE) of frog photoreceptor membranes has been assayed by measuring the evolution of protons that accompanies cyclic GMP hydrolysis. The validity of this assay has been confirmed by comparison with an isotope assay used in previous studies (Robinson et al. 1980. J. Gen. Physiol. 76: 631-645). The PDE activity elicited by either flash or continuous dim illumination is reduced if ATP is added to outer segment suspensions. This desensitization is most pronounced at low calcium levels. In 10(-9) M Ca++, with 0.5 mM ATP and 0.5 mM GTP present, PDE activity remains almost constant as dim illumination and rhodopsin bleaching continue. At intermediate Ca++ levels (10-7-10-5M) the activity slowly increases during illumination. Finally, in 10(-4) and PDE activity is more a reflection of the total number of rhodopsin molecules bleached than of the rate of the rhodopsin bleaching. At intermediate or low calcium levels a short-lived inhibitory process is revealed by observing a nonlinear summation of responses of the enzyme to closely spaced flashes of light. Each flash makes PDE activity less responsive to successive flashes, and a steady state is obtained in which activation and inactivation are balanced. It is suggested that calcium and ATP regulation of PDE play a role in the normal light adaption processes of frog photoreceptor membranes.     

Molecular design of an amplification cascade in vision

The photoexcitation of rhodopsin triggers a cascade that results in the hydrolysis of a large number of molecules of cyclic GMP. The molecular mechanism of this amplification cascade has been delineated. Transducin, a multisubunit perpheral membrane protein, is the information-carrying intermediate in the activation of the cyclic GMP phosphodiesterase. Photoexcited rhodopsin (R*) castalyzes the exchange of GRP for GDP bound to the α-subunit of transducin (T). About 500 molecules of T_α-GTP are formed per absorbed photon at low light levels. T_α-GTP, rekeased from the β- and γ-subunits of transducin, then activates the phosphodiesterase by relieving an inhibitory constraint imposed by its small sununit. Each actived phosphodiesterase molecule hydrolyzes more than 100 cyclic GMP/s, giving an overall gain of more than 500,000. Photoexcited rhodopsin triggers the activation of a molecule of transducin in a millisecond, which is sufficiently rapid to enable this cascade to participate in visual excitation. Hydrolysis of GTP bound to T_α seves to restore the system to the dark state. Transducin, like the G proteins of the adenylate cyclase casecade, can be specifically ADP-ribosylated by cholera toxin and pertussis toxin. In both cascades, labling by pertussis toxin blocks the capacity of transducin to interact with the excited receptor, whereas labeling by cholera toxin inhibits the hydrolysis of bound GTP, leading to persistent activation. Moreover, the moleculaar design of the hormone-triggered cyclic AMP cascade is similar to that of the light-triggered cyclic GMP cascade. It seems likely that transducin, the stimulatory G protein, the inhibitor G protein, and the ras protein are members of the same family of signal amplifiers. The study of the cyclic nucleotide cascade of vision is providing rewarding views of recurring motifs of signal amplification in nature.     

Cyclic nucleotide phosphodiesterases associated with bovine retinal outer-segment fragments.

ATP-dependent cyclic GMP phosphodiesterase activity (EC 3.1.4.16) associated with bovine retinal outer-segment fragment preparations was stimulated an order of magnitude by light, confirming the results of Miki et al. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3820-3824 at Yale for the frog system. In contrast to the results of the Yale group, however, light stimulation was not observed for cyclic AMP as substrate. A direct relationship of bovine rhodopsin bleaching to phosphodiesterase activation differs from a previous report by the Yale group that full activation of the frog enzyme was achieved by bleaching of a maximum of 2% rhodopsin. Phosphodiesterase activity could be qualitatively removed from the fresh outer-segment preparations with isotonic sucrose which apparently did not disrupt the plasmalemma or discs. Activity recovered from the washing was not light sensitive. Two Km values were determined for cyclic AMP, 5 and 0.05 mM; for cyclic GMP a Km of 0.22 mM was found. All Km values were determined in the presence of 1 mM ATP in the dark. Sonication of fresh outer segments or storing at -20 degrees C abolished the light response. However, storage at -76 degrees C fully preserved it.     

A light-stimulated increase of cyclic GMP in squid photoreceptors

Photoreceptor outer segments isolated from squid retina are known to contain a light-activated GTP-binding protein. Here it is shown that these photoreceptors contain around 0.01 mol cyclic GMP per mol rhodopsin. Adding GTP in the dark stimulates the production of 0.0003-0.001 mol cyclic GMP/mol rhodopsin per min. GTP and light cause a 2-fold faster increase in cyclic GMP. These results show that either (1) squid rhodopsin activates a guanylate cyclase, or (2) there is a constant guanylate cyclase activity and photoexcited rhodopsin inhibits a cyclic GMP phosphodiesterase.     

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.     

Influence of light and calcium on guanosine 5'-triphosphate in isolated frog rod outer segments.

Frog rod outer segments contain approximately 0.25 mol of GTP and 0.25 mol of ATP per mol of rhodopsin 3 min after their isolation from the retina. UTP and CTP are present at 10-fold and 100-fold lower levels, respectively. Concentrations of GTP and ATP decline in parallel over the next 4 min to reach relatively stable levels of 0.1 mol per mol of rhodopsin. Illumination reduces the concentration of endogenous GTP but not ATP. This light-induced decrease in GTP can be as large as 70% and has a half-time of 7 s. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity, but partially returns to its dark-adapted level after brief illumination. The magnitude of the decrease increases as a linear function of the logarithm of continuous light intensity at levels which bleach between 5 X 10(2) and 5 X 10(6) rhodopsin molecules/outer segment per second. This exceeds the range of intensities over which illumination causes decreases in the cyclic GMP content and permeability of isolated outer segments (Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). Thus, over 4 log units of light intensity, a sensitivity control mechanism functions to make extended illumination less effective in stimulating a GTP decrease. GTP levels in dark-adapted outer segments are sensitive to changes in calcium concentration in the suspending medium. If the external calcium concentration is reduced to 10(-8) M, GTP concentration is lowered to the same level caused by saturating illumination, and the GTP remaining is no longer light-sensitive. Lowering calcium concentration to intermediate levels between 10(-6) and 10(-8) M reduces GTP to stable intermediate levels, and the GTP remaining can be reduced by light. Restoration of millimolar calcium drives synthesis of GTP, but not of ATP, and GTP lability towards illumination is again observed. These calcium-induced changes in GTP are diminished by the addition of the divalent cation ionophore A23187. Lowering or raising magnesium levels does not influence the GTP concentration. These data raise the possibility that light activates either a calcium transport mechanism driven by the hydrolysis of GTP, or some other calcium-sensitive GTPase activity of unknown function. Known light-dependent reactions involving cyclic nucleotide transformations and rhodopsin phosphorylation appear to account for only a small fraction of the light-induced GTP decrease.     

Cyclic GMP and photoreceptor function.

A single photon can be detected by a rod photoreceptor cell. The absorption of light by rhodopsin triggers a cascade of reactions that amplifies the photon signal and results in ion channel closure with hyperpolarization of the rod photoreceptor cell. Light-induced conformational changes in rhodopsin facilitate the binding of a guanosine nucleotide-binding protein, transducin, which then undergoes a GTP-GDP exchange reaction and dissociation of the transducin complex. A subunit of transducin then activates a phosphodiesterase complex that hydrolyzes cyclic GMP. In darkness, cyclic GMP binds to cation channels of the photoreceptor plasma membrane, maintaining them in an open configuration. The light-induced reduction in cyclic GMP concentration dissociates the bound cyclic GMP, resulting in channel closure and hyperpolarization. Down-regulation of the cascade involves other proteins that block the interaction of transducin with rhodopsin and another protein that may interfere with transducin recycling. Cone photoreceptors possess a light-activated cascade that follows the rod format, but it is composed of proteins that are homologous to those of rod photoreceptors. Phototransduction in invertebrate photoreceptors uses rhodopsin to activate a cascade that uses phosphoinositides and calcium ion to regulate membrane polarization.     

Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation

Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin.     

Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments.

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.