Protein-lipid interactions at membrane surfaces: a deuterium and phosphorus nuclear magnetic resonance study of the interaction between bovine rhodopsin and the bilayer head groups of dimyristoylphosphatidylcholine

Rhodopsin, isolated from bovine retinal rod outer segment disk membranes, has been reconstituted into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine which was deuterated in the terminal methyl groups of the choline polar head group. By use of a mixed detergent system of cholate and octyl glucoside to solubilize the phospholipid and rhodopsin, 15 membrane complexes of predetermined phospholipid to rhodopsin mole ratios of between 350:1 and 65:1 have been produced by exhaustive dialysis and studied by a variety of techniques. Electron micrographs of replicas from freeze-fractured membrane complexes showed that the majority of the lipid, for all rhodopsin:phospholipid ratios, was contained in large bilayer vesicles with diameters in excess of 400 nm. Complexes produced with rhodopsin from frozen retina produced an absorption maximum at 478 nm after photobleaching whereas rhodopsin from fresh retina could be bleached more completely to an absorption maximum at 380 nm. Deuterium nuclear magnetic resonance (NMR) spectra from the lipid head groups of bilayers above the gel to liquid-crystalline phase transition temperature were shown to be sensitive in a systematic way to the presence of rhodopsin which could be bleached to 380 nm. The measured quadrupole splittings, taken as the separation of the turning points of the recorded NMR spectra, decreased from a value of 1.28 kHz for protein-free bilayers to approximately 0.40 kHz for bilayers containing 65 molecules of phospholipid for each rhodopsin at 32 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhodopsin in dimyristoylphosphatidylcholine-reconstituted bilayers forms metarhodopsin II and activates Gt

The photochemical intermediate metarhodopsin II (meta II; lambda max = 380 nm) is generally identified with rho*, the conformation of photolyzed rhodopsin which binds and activates the visual G-protein, Gt [Emeis, D., & Hoffman, K.P. (1981) FEBS Lett. 136, 201-207]. Purified bovine rhodopsin was incorporated into vesicles consisting of dimyristoylphosphatidylcholine (DMPC), and the rapid formation of a photochemical intermediate absorbing maximally at 380 nm was quantified via both flash photolysis and equilibrium spectral measurements. Kinetic and equilibrium spectral measurements performed above the Tm of DMPC showed that Gt, in the absence of GTP, enhances the production of the 380-nm-absorbing species while reducing the concentration of the 478-nm-absorbing species, metarhodopsin I (meta I), in a manner similar to that observed in the native rod outer segment disk membrane. This Gt-induced shift in the equilibrium concentration of photointermediates indicated that the species with an absorbance maximum at 380 nm was meta II. The presence of rho* in the DMPC bilayer was established via measurements of photolysis-induced exchange of tritiated GMPPNP, a nonhydrolyzable analogue of GTP, on Gt. Above Tm, the metarhodopsin equilibrium is strongly shifted toward meta I relative to the native rod outer segment disk membrane; however, at 37 degrees C, 40% of the photointermediates are in the form of meta II. The formation of meta II above Tm is slowed by a factor of ca. 2 relative to the disk membrane. Below Tm, the equilibrium is shifted still further toward meta I, and meta II forms ca. 7 times slower than in the disk membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein rotational diffusion and lipid/protein interactions in recombinants of bovine rhodopsin with saturated diacylphosphatidylcholines of different chain lengths studied by conventional and saturation-transfer electron spin resonance.

Bovine rhodopsin has been reconstituted in seven different saturated diacylphosphatidylcholine species of odd and even chain lengths from C-12 to C-18 at a lipid/protein ratio (60:1 mol/mol) comparable to that in the native rod outer segment disk membrane. All recombinants were found to be photochemically active, in that optical bleaching produced a temperature- and lipid chain-length-dependent mixture of species absorbing at 480 and 380 nm. Both the rotational diffusion of rhodopsin and lipid-protein interactions in the various recombinants were studied by saturation transfer and conventional electron spin resonance spectroscopy of spin-labeled rhodopsin and of spin-labeled phosphatidylcholine, respectively. In the fluid lipid phase, the rotational diffusion rate of rhodopsin was found to be dependent on the lipid chain length of the different recombinants in a nonmonotonic manner. The diffusion rate in dilauroylphosphatidylcholine was found to be very slow, indicating extensive protein aggregation, whereas that in dipentadecanoylphosphatidylcholine was rapid (effective correlation time ca. 7 microseconds), consistent with the presence of monomeric protein. For recombinants with longer lipid chain lengths, the rotational diffusion rate again decreased, indicating the presence of di- or oligomeric protein. The fraction of lipid motionally restricted at temperatures in the fluid phase was also dependent on the chain length of the phosphatidylcholine used in the reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation of stable cattle rod outer segments with an intact plasma membrane.

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter. Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH. The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

The isolation of stable cattle rod outer segments with an intact plasma membrane

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter.Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH.The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Reconstitution of an electrically active conformational transition in rhodopsin-containing membranes

An electrically active event that has been observed in native rod outer segment disk membranes can be reconstituted into membrane vesicles containing purified rhodopsin and defined phospholipids. The magnitude of this charge-transfer event, as estimated using spin-labeled derivatives of hydrophobic ions, is a function of the phospholipid composition. In reconstituted membranes containing rhodopsin and egg phosphatidylcholine, the charge transferred during this event is approximately 10% that measured in the native system. The addition of 20 mol% egg phosphatidylethanolamine, phosphatidic acid or brain phosphatidylserine returns the magnitude of the charge transfer to within 60 to 100% of the native activity. The response seen in the reconstituted membrane system is consistent with a previously proposed interfacial charge-transfer mechanism.     

Some quantitative characteristics of the frog retinal rod outer segments]

Retinal rod outer segments in frogs were studied by means of light microscopy, refractometry, microspectrophotometry, and electron microscopy. Analysis of the data obtained shows that an unidentified substance, which makes up about 50% of outer segment dry weight, is lost during routine biochemical investigations. The protein parts of the rhodopsin molecules make up 85% of the outer segments proteins and 25% of outer segment dry weight. Rhodopsin molecules can be arranged in a square array with a unit cell side of about 7 nm on one side of each disk membrane. Lipids in a single membrane occupy only 2 nm, and disk membranes are strongly hydrated.     

Rhodopsin photointermediates in two-dimensional crystals at physiological temperatures

Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystal suspensions were studied by measuring the time-dependent absorbance changes produced after excitation with 7 ns laser pulses at 15, 25, and 35 degrees C. The crystalline environment favored the Meta I(480) photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 degrees C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I(480) decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35 degrees C and quenched to 19 degrees C showed that a rapid equilibrium existed on the approximately 1 s time scale, which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII(a)) forms in 2D rhodopsin crystals but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium.

The effect of phospholipid bilayer acyl chain packing free volume on the equilibrium concentration of the form of photolyzed rhodopsin which initiates visual signal transduction, metarhodopsin II (meta II), is examined in reconstituted systems formed from the saturated phospholipid dimyristoylphosphatidylcholine (DMPC) and in the polyunsaturated phospholipid sn-1-palmitoyl-sn-2-arachidonoylphosphatidylcholine (PAPC) with and without 30 mol% cholesterol. The extent of meta II formation is determined from both flash photolysis measurements and rapidly acquired absorbance spectra. Equilibrium and dynamic properties of the lipid bilayer are characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH orientational properties are characterized by fv, a parameter which reflects the volume available for probe reorientation in the bilayer, relative to that available in an unhindered, isotropic environment [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5121-5126]. The metarhodopsin I in equilibrium with meta II equilibrium constant, Keq has a linear relationship with fv for rhodopsin in PAPC vesicles with and without cholesterol as well as for rhodopsin in DMPC vesicles, and these two correlation lines have different slopes. The correlations between Keq and fv in PAPC and DMPC systems are compared with a similar correlation in the native rod outer segment disk membrane and one reported previously in an egg phosphatidylcholine (egg PC) system [Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990) Biochemistry 29, 9143-9149].(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural localization of rhodopsin in the vertebrate retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.     

Differential Rhodopsin Regeneration in Photoreceptor Membranes is Correlated with Variations in Membrane Properties

Rhodopsin, the major transmembrane protein in both the plasma membrane and the disk membranes of photoreceptor rod outer segments (ROS) forms the apo-protein opsin upon the absorption of light. In vivo the regeneration of rhodopsin is necessary for subsequent receptor activation and for adaptation, in vitro this regeneration can be followed after the addition of 11-cis retinal. In this study we investigated the ability of bleached rhodopsin to regenerate in the compositionally different membrane environments found in photoreceptor rod cells. When 11-cis retinal was added to bleached ROS plasma membrane preparations, rhodopsin did not regenerate within the same time course or to the same extent as bleached rhodopsin in disk membranes. Over 80% of the rhodopsin in newly formed disks regenerated within 90 minutes while only 40% regenerated in older disks. Since disk membrane cholesterol content increases as disks are displaced from the base to the apical tip of the outer segment, we looked at the affect of membrane cholesterol content on the regeneration process. Enrichment or depletion of disk membrane cholesterol did not alter the % rhodopsin that regenerated. Bulk membrane properties measured with a sterol analog, cholestatrienol and a fatty acid analog, cis parinaric acid, showed a more ordered, less fluid, lipid environment within plasma membrane relative to the disks. Collectively these results show that the same membrane receptor, rhodopsin, functions differently as monitored by regeneration in the different lipid environments within photoreceptor rod cells. These differences may be due to the bulk properties of the various membranes.     

The study of photoconduction of artificial lipid membranes incorporating rhodopsin. The simultaneous changes of membrane conduction and rhodopsin fluorescence

The protein fluorescence changes of rod outer segment fragments during bleaching were studied. Flash caused a fluorescence intensity drop by about 6%. The time constant of this process was30 msec and coincided with the time constant of increasing the permeability of an artificial lipid membrane containing rhodopsin and of Metarhodopsin I decay. In the presence of hydroxylamine the fluorescence intensity increases after the initial drop. The second process time constant was about 300 msec and coincided with the conduction drop time constant of the artificial membrane containing rhodopsin. A new intermediate — Metarhodopsin II₁ is proposed. It has the Metarhodopsin II absorption spectrum, lives for about 300 msec at room temperature, does not react with hydroxylamine, and increases the permeability of a disk membrane.     

Distribution of charge on photoreceptor disc membranes and implications for charged lipid asymmetry.

A novel spin labeling technique is used to determine both the inner and outer surface potentials of isolated rod outer segment disc membranes and of reconstituted membranes containing rhodopsin with defined lipid compositions. It is shown that these potentials can be accounted for in a consistent manner by the accepted model of rhodopsin, the known lipid composition, and the Gouy-Chapman theory, provided the charged lipid is asymmetric in the membrane, with approximately 75% on the external surface.     

Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. Several control experiments indicated that the labeled proteins are integral membrane proteins and that label is limited to the plasma membrane. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger.     

Orientation of the rhodopsin sugar moiety in bovine disk membrane.

Rhodopsin from the bovine rod outer segment contains a covalently linked carbohydrate moiety (Heller, J. & Lawrence, M.A. (1973) Biochemistry 9, 864--868). We studied the location of this carbohydrate moiety on the disk membrane by using ferritin-conjugated concanavalin A and concanavalin A labelled with fluorescein isothiocyanate. Electron microscopic observation of sonicated disk membrane that was labelled with ferritin-concanavalin A revealed the electron-dense image of ferritin on the inner surface of the disk membrane and not on its outer surface. Intact disk membrane that was similarly treated with ferritin-concanavalin A showed a complete absence of ferritin molecules on its surface. In an independent series of experiments we confirmed that the sonicated disk membrane bound three to five times more fluorescein-labelled concanavalin A than the intact disk membrane did. From these experiments we conclude that the carbohydrate moiety of bovine rhodopsin is located on the inner surface of the disk membrane, in agreement with the report by Rohlich on the frog rod outer segment disk membrane (Rohlich, P. (1976) Nature 263, 789--791).     

Lipid-protein interactions mediate the photochemical function of rhodopsin

We have investigated the molecular features of recombinant membranes that are necessary for the photochemical function of rhodopsin. The magnitude of the metarhodopsin I to metarhodopsin II phototransient following a 25% +/- 3% bleaching flash was used as a criterion of photochemical activity at 28 degrees C and pH 7.0. Nativelike activity of rhodopsin can be reconstituted with an extract of total lipids from rod outer segment membranes, demonstrating that the protein is minimally perturbed by the reconstitution protocol. Rhodopsin photochemical activity is enhanced by phosphatidylethanolamine head groups and docosahexaenoyl (22:6 omega 3) acyl chains. An equimolar mixture of phosphatidylethanolamine and phosphatidylcholine containing 50 mol% docosahexaenoyl chains results in optimal photochemical function. These results suggest the importance of both the head-group and acyl chain composition of the rod outer segment lipids in the visual process. The extracted rod lipids and those lipid mixtures favoring the conformational change from metarhodopsin I to II can undergo lamellar (L alpha) to inverted hexagonal (HII) phase transitions near physiological temperature. Interaction of rhodopsin with membrane lipids close to a L alpha to HII (or cubic) phase boundary may thus lead to properties which influence the energetics of conformational states of the protein linked to visual function.     

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles.

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.     

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.