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.     

Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation

In the vertebrate rod outer segment (ROS), the light-dependent activation of a GTP-binding protein (G-protein) and phosphodiesterase (PDE) is quenched by a process that requires ATP [Liebman, P.A., & Pugh, E.N. (1979) Vision Res. 19, 375-380]. The ATP-dependent quenching mechanism apparently requires the phosphorylation of photoactivated rhodopsin (Rho*); however, a 48-kilodalton protein (48K protein) has also been proposed to participate in the inactivation process. Purified species of phosphorylated rhodopsin containing 0, 2, or greater than or equal to 4 (high) phosphates per rhodopsin (PO4/Rho) were reconstituted into phosphatidylcholine (PC) vesicles and reassociated with a hypotonic extract from isotonically washed disk membranes that were depleted of 48K protein; PDE activation, in response to bleaching from 0.01% to 15% of the rhodopsin present, was measured. PDE activity was reduced by at least 30% at high fractional rhodopsin bleaches and by greater than 80% at low fractional rhodopsin bleaches in high PO4/Rho samples when compared to the activity measured in O PO4/Rho controls. A phosphorylation level of 2 PO4/Rho produced PDE activities that were intermediate between O PO4/Rho and high PO4/Rho samples at low bleaches, but were identical with the O PO4/Rho samples at high rhodopsin bleaches. Rhodopsin phosphorylation is thus capable of producing a graded inhibition of light-stimulated PDE activation over a limited range of (near physiological) bleach levels. This effect become less pronounced as the bleach levels approach those that saturate PDE activation. These results are consistent with increasing levels of phosphorylation, producing a reduction of the binding affinity of G-protein for Rho*.     

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.     

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.     

ATP can promote activation and deactivation of the rod cGMP-phosphodiesterase. Kinetic light scattering on intact rod outer segments

The AT (amplified transient) signal is a flash-induced increase of the near-infrared light scattering from isolated bovine rod outer segments and is interpreted as a monitor of cGMP-phosphodiesterase activation [(1985) FEBS Lett. 188, 15-20]. We have investigated the effects of ATP and cyclic GMP on this signal. It has been found that ATP enhances the AT signal, the relative effect being the largest for low photoexcitation (approximately 1 rhodopsin per disc membrane). At a high rhodopsin turnover, which saturates the AT amplitude, the effect of ATP is to accelerate the rise of the signal. ATP can also accelerate the falling phase of the signal. This deactivating effect depends on the simultaneous presence of cyclic GMP. The results indicate that ATP acts on the phosphodiesterase activation cycle, promoting activation as well as deactivation, dependent on cGMP as a cofactor.     

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.     

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)     

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.     

A link between rhodopsin and disc membrane cyclic nucleotide phosphodiesterase. Action spectrum and sensitivity to illumination.

Frog (Rana pipiens) rod outer segment disc membranes contain guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1.c) which, in the presence of ATP, is stimulated 5- to 20-fold by illumination. The effectiveness of monochromatic light of different wavelengths in activating phosphodiesterase was examined. The action spectrum has a maximum of 500 nm, and the entire spectrum from 350 to 800 nm closely matches the absorption spectrum of rhodopsin, which is apparently the pigment which mediates the effects of light on phosphodiesterase activity. trans-Retinal alone does not mimic light. Half-maximal activation of the phosphodiesterase occurs with a light exposure which bleaches 1/2000 of the rhodopsins. Half-maximal activation can also be achieved by mixing 1 part of illuminated disc membranes in which the rhodopsin is bleached with 99 parts of unilluminated membranes. Regeneration of bleached rhodopsin by addition of 11-cis-retinal is illuminated disc membranes reverses the ability of these membranes to activate phosphodiesterase in unilluminated membranes. If the rhodopsin regenerated by 11-cis-retinal is illuminated again, it regains the ability to activate phosphodiesterase. These studies show that the levels of cyclic nucleotides in vetebrate rod outer segments are regulated by minute amounts of light and clearly indicate that rhodopsin is the photopigment whose state of illumination is closely linked to the enzymatic activity of disc membrane phosphodiesterase.     

Light-induced dephosphorylation of two proteins in frog rod outer segments: influence of cyclic nucleotides and calcium

Two minor proteins of frog rod outer segments become phosphorylated when retinas are incubated in the dark with 32Pi. The proteins, designated component I (13,000 daltons) and component II (12,000 daltons), are dephosphorylated when retinas are illuminated. The dephosphorylation is reversible; the two proteins are rephosphorylated when illumination ceases. Each outer segment contains approximately 10(6( molecules of components I and II. These remain associated with both fragmented and intact outer segments but dissociate from the outer segment membranes under hypoosmotic conditions. The extent of the light-induced dephosphorylation increases with higher intensities of illumination and is maximal with continuous illumination which bleaches 5.0 x 10(5) rhodopsin molecules/outer segment per second. Light which bleaches 5.0 x 10(3) rhodopsin molecules/outer segment per second causes approximately half-maximal dephosphorylation. This same intermediate level of illumination causes half-suppression of the light-sensitive permeability mechanism in isolated outer segments (Brodie and Bownds. 1976. J. Gen Physiol. 68:1-11) and also induces a half-maximal decrease in their cyclic GMP content (Woodruff et al. 1977. J. Gen. Physiol. 69:667-679). The phosphorylation of components I and II is enhanced by the addition of cyclic GMP or cyclic AMP to either retinas or isolated rod outer segments maintained in the dark. Several pharmacological agents which influence cyclic GMP levels in outer segments, including calcium, cause similar effects on the phosphorylation of components I and II and outer segment permeability. Although the cyclic nucleotide-stimulated phosphorylation can be observed either in retinas or isolated rod outer segments, the light-induced dephosphorylation is observed only in intact retinas.     

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.     

Binding and activation of rod outer segment phosphodiesterase and guanosine triphosphate binding protein by disc membranes: influence of reassociation method and divalent cations

Attempts to optimize the recovery of light-stimulated phosphodiesterase activity following reassociation of the hypotonically extractable proteins derived from retinal rod segments with hypotonically stripped disc membranes lead to the following observations: the best reassociations were obtained by mixing proteins and stripped disc membranes under hypotonic conditions and slowly increasing the salt concentration; the binding of G-protein and phosphodiesterase to stripped disc membrane occurs in less than 5 minutes and the recovery of light-stimulated phosphodiesterase activation in response to subsaturating stimulus levels requires 2-3 h to plateau. Stripped disc membranes and proteins were reassociated in 'isotonic' buffers containing KCl/NaCl, KCl/NaCl plus Mg2+, or KCl/NaCl plus Ca2+. Large fractional rhodopsin bleaches produced nearly identical light-stimulated phosphodiesterase activities in each of these samples and in the control rod outer segment membranes. Rod outer segment membranes and reassociated stripped disc membrane samples containing divalent cations showed similar phosphodiesterase activities in response to low fractional rhodopsin bleaches (e.g. less than or equal to 0.1%), however, samples devoid of divalent cations during reassociation required rhodopsin bleaches up to 10-fold larger to elicit comparable phosphodiesterase activities. These results suggest that not all phosphodiesterase and/or G-protein molecules bound to the disc membrane surface are equivalent with regard to their efficiency of activation by bleached rhodopsin and that divalent cations can modulate the distribution of G-protein and/or phosphodiesterase between these populations.     

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.     

Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin)

The inactivation of excited rhodopsin in the presence of ATP, rhodopsin kinase, and/or arrestin has been studied from its effect on the two subsequent steps in the light-induced enzymatic cascade: metarhodopsin II catalyzed activation of G-protein and G-protein-dependent activation of cGMP phosphodiesterase. The inactivation of G-protein (from light-scattering measurements) and that of phosphodiesterase (from measurements of cGMP hydrolysis) have been studied and compared in reconstituted systems containing various combinations of the proteins involved (rhodopsin, G-protein, phosphodiesterase, kinase, and arrestin). Our results show that rhodopsin kinase alone can terminate the activation of G-protein and that arrestin speeds up the process at a relative concentration similar to that reported in the rod (half-maximal effect at 50 nM for 4.4 microM rhodopsin). Measurements of rhodopsin phosphorylation under identical conditions show that in the presence of arrestin total metarhodopsin II inactivation is achieved when only 0.5-1.4 phosphates are bound per bleached rhodopsin, whereas in the absence of arrestin it requires binding of 12-16 phosphates per bleached rhodopsin. Phosphodiesterase activity can similarly be turned off by kinase, and the process is similarly accelerated by arrestin.     

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.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

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.     

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.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat heart. Solubilization and characterization of a single enzymatic form

Approximatively 2–8% of the cyclic nucleotide phosphodiesterase activity of a crude 1000 g supernatant from rat heart was associated with the washed 105,000 g pellet fraction. This activity exhibited biphasic Lineweaver-Burk plots over a large range of cyclic nucleotides concentrations. Concave-Bownward plots were obtained with cyclic AMP as the assay substrate, while cyclic GMP gave rise to concave-upward plots. Treatment of this particulate fraction by freezing and thawing and then with 2% Lubrol PX released the major part of phosphodiesterase activity into the supernatant (70 and 90% for cyclic AMP and cyclic GMP phosphodiesterase activities respectively). Isoelectric focusing of the solubilized enzyme revealed a single peak of phosphodiesterase activity. While the Lineweaver-Burk plots of cyclic AMP phosphodiesterase activity were not markedly modified by detergent treatment kinetic plots of cyclic GMP phosphodiesterase activity underwent a drastic transformation during the overall solubilization procedure. The substantial increase in the cyclic GMP rate of hydrolysis observed at low substrate level might explain the difference in the apparent yield of solubilization between cyclic AMP and cyclic GMP phosphodiesterase activities.