Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.     

Control of the cyclic GMP phosphodiesterase of frog photoreceptor membranes

The light-activated cyclic GMP phosphodiesterase (PDE) of frog photoreceptor membranes has been assayed in isolated outer segments suspended in a low-calcium Ringer's solution. Activation occurs over a range of light intensity that also causes a decrease in the permeability, cyclic GMP levels, and GTP levels of isolated outer segments. At intermediate intensities, PDE activity assumes constant intermediate values determined by the rate of rhodopsin bleaching. Washing causes an increase in maximal enzyme activity. Increasing light intensity from darkness to a level bleaching 5 x 10(3) rhodopsin molecules per outer segment per second shifts the apparent Michaelis constant (Km) from 100 to 900 microM. Maximum enzyme velocity increases at least 10-fold. The component that normally regulates this light- induced increase in the Km of PDE is removed by the customary sucrose flotation procedures. The presence of 10(-3) M Ca++ increases the light sensitivity of PDE, and maximal activation is caused by illumination bleaching only 5 x 10(2) rhodopsin molecules per outer segment per second. Calcium acts by increasing enzyme velocity while having little influence on Km. The effect of calcium appears to require a labile component, sensitive to aging of the outer segment preparation. The decrease in the light sensitivity of PDE that can be observed upon lowering the calcium concentration may be related to the desensitization of the permeability change mechanism that occurs during light adaptation of rod photoreceptors.     

Control of light-activated phosphorylation in frog photoreceptor membranes.

In this paper, we examine some factors which regulate the efficiency of light in activating rhodopsin phosphorylation. We have measured phosphate incorporation after illumination in suspensions of bullfrog rod outer segments incubated with [gamma-32P]ATP. We observed that delaying ATP addition after illumination causes maximum phosphate incorporation to decrease 80% within 2 h. This decay occurs in urea-treated, extracted rod outer segment membranes. The decay of the light effect is not influenced by regeneration of opsin to rhodopsin or the presence of long-lived photoproducts. However, regeneration of opsin increases the amount of phosphorylation initiated by a second exposure to light. Further phosphorylation can also occur after phosphate groups have been removed from the membranes by dephosphorylation. Finally, we have confirmed our earlier observation that small amounts of light (bleaching less than 5% of the rhodopsin present) are more effective, by tenfold, in initiating phosphorylation than are larger amounts.     

Axial gradients of rhodopsin in light-exposed retinal rods of the toad

Exposure of an intact vertebrate eye to light bleaches the rhodopsin in the photoreceptor outer segments in spatially nonuniform patterns. Some axial bleaching patterns produced in toad rods were determined using microspectrophotometric techniques. More rhodopsin was bleached at the base of the outer segment than at the distal tip. The shape of the bleaching gradient varied with the extent of bleach and with the spectral content of the illuminant. Monochromatic light at the lambda max of the rhodopsin gave rise to the steepest bleaching gradients and induced the greatest changes in the form of the gradient with increasing extent of bleach. These results were consistent with a mathematical model for pigment bleaching in an unstirred sample. The model did not fit bleaching patterns resulting from special lighting conditions that promoted the photoregeneration of rhodopsin from the intermediates of bleaching. Prolonged light adaptation of toads could also produce axial rhodopsin gradients that were not fit by the bleaching model. Under certain conditions the axial gradient of rhodopsin in a rod outer segment reversed with time in the light: the rhodopsin content became highest at the base. This result could be explained by an interaction between the pattern of bleaching and the intracellular topography of regeneration.     

Measurement of fast light-induced disc shrinkage within bovine rod outer segments by means of a light-scattering transient

A fast light-induced light-scattering transient, previously found in rod outer segment suspension, the so-called P-signal (Hofmann, K.P., Uhl, R., Hoffmann, W. and Kreutz, W. (1976) Biophys. Struct. Mechanism 2, 61–77), is described in more detail.The effect has the same action spectrum as rhodopsin bleaching. It is not regenerated with 11-cis retinal.The response is not linear with light-intensity for flashes which bleach more than 2.0% of rhodopsin; it saturates at an intensity corresponding to 15% rhodopsin bleaching.The wavelength- and scattering angle dependence lead to the conclusion that the change in light-scattering reflects a shrinkage of an osmotic compartment of the rod outer segment.The only compartment which we found to be intact in our rod outer segment preparations was the disc or rod sac; therefore, the effect must be attributed to a light-induced shrinkage of the rhodopsin-containing disc organelles.The overall effect (15% of rhodopsin is bleached) is in the range of 0.5–1.5% of the original volume.A light-induced passive cation-efflux from the disc, e.g. of Ca²⁺, can be ruled out as a possible molecular origin of the disc-shrinkage in our preparations.     

Biochemical aspects of the visual process. XXXVIII. Effects of lateral aggregation on rhodopsin in phospholipase C-treated photoreceptor membranes

Treatment of bovine rod outer segments with phospholipase C leads to largely lipid-depleted membranous structures. Under these conditions rhodopsin remains spectrally intact, but its thermal stability and regeneration capacity are decreased, whereas upon illumination the metarhodopsin I to II transition is blocked. These observations can be explained on the basis of the previously demonstrated lateral aggregation of rhodopsin molecules which, on the one hand leads to a (partial) shielding of these molecules and, on the other hand, might impose constraints on the flexibility of the molecule to undergo light-induced conformational changes.Upon reconstitution of these lipid-depleted preparations with amphipathic lipids by means of a detergent dialysis procedure, the aggregates are apparently rearranged to lipid bilayer structures with complete recovery of the original rhodopsin properties. Under our conditions the nature of the polar head groups and the fatty acids is not critical in this respect. Simple addition of amphipathic lipids, without the use of detergent, restores the rhodopsin properties only in the case of rod outer segment lipids and of didecanoylphosphatidylcholine, and even then only occasionally.These results are discussed in the light of the strong analogy in properties between phospholipase C-treated rod outer segment membranes and lipid- and detergent-free rhodopsin obtained by affinity chromatography. It is concluded that rhodopsin must be in a freely dispersed state in order to function properly. Apparently, a non-specific lipid bilayer fulfills this condition for the regeneration capacity, whereas normal photolytic behaviour requires, in addition, a minimal membrane fluidity according to the observations of other investigators. Presumably, the uniquely high phospholipid unsaturation of rod outer segment membranes is important for another, as yet unassessed, function of rhodopsin or the photoreceptor membrane.     

Amplitude, kinetics, and reversibility of a light-induced decrease in guanosine 3'',5''-cyclic monophosphate in frog photoreceptor membranes

The concentration of guanosine 3',5'-cyclic monophosphate (cyclic GMP) has been examined in suspensions of freshly isolated frog rod outer segments using conditions which previously have been shown to maintain the ability of outer segments to perform a light-induced permeability change (presence of calf serum, anti-oxidant, and low calcium concentration). Illumination causes a rapid decrease in cyclic GMP levels which has a half-time approximately 125 ms. With light exposures that bleach less than 100 rhodopsin molecules in each rod outer segment, at least 10(4)-10(5) molecules of cyclic GMP are hydrolyzed for each rhodopsin molecule bleached. Half of the total cyclic GMP in each outer segment, approximately 2 X 10(7) molecules, is contained in the light-sensitive pool. If outer segments are exposed to continuous illumination, using intensities which bleach between 5.0 X 10(1) and 5.0 X 10(4) rhodopsin molecules/outer segment per second, cyclic GMP levels fall to a value characteristic for the intensity used. This suggests that a balance between synthesis and degradation of cyclic GMP is established. This constant level appears to be regulated by the rate of bleaching rhodopsin molecules (by the intensity of illumination), not the absolute number of rhodopsin molecules bleached...     

Recoverin and rhodopsin kinase activity in detergent-resistant membrane rafts from rod outer segments

Cholesterol-rich membranes or detergent-resistant membranes (DRMs) have recently been isolated from bovine rod outer segments and were shown to contain several signaling proteins such as, for example, transducin and its effector, cGMP-phosphodiesterase PDE6. Here we report the presence of rhodopsin kinase and recoverin in DRMs that were isolated in either light or dark conditions at high and low Ca2+ concentrations. Inhibition of rhodopsin kinase activity by recoverin was more effective in DRMs than in the initial rod outer segment membranes. Furthermore, the Ca2+ sensitivity of rhodopsin kinase inhibition in DRMs was shifted to lower free Ca2+ concentration in comparison with the initial rod outer segment membranes (IC50=0.76 microm in DRMs and 1.91 microm in rod outer segments). We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-beta-cyclodextrin yielded a similar shift of the Ca2+-dependent dose-response curve of rhodopsin kinase inhibition. Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form. These data suggest that the Ca2+-dependent feedback loop that involves recoverin is spatially heterogeneous in the rod cell.     

Biochemical aspects of the visual process. XXVIII. Classification of sulfhydryl groups in phodopsin and other photoreceptor membrane proteins.

Reaction of isolated bovine rod outer segment membrane with radioactive N-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule. On the basis of their reactivity towards rho-chloromercuribenzoate and rho-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with the rho-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and isprobably located in the vicinity of the chromophoric center. The differences between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) or N-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of the rho-chloromercuri-derivatives.     

All-trans retinol in rod photoreceptor outer segments moves unrestrictedly by passive diffusion

The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption. Subsequently, all-trans retinal is released from the protein and reduced to all-trans retinol, the first step in the recycling of rhodopsin's chromophore group through the series of reactions that constitute the visual cycle. The concentration of all-trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all-trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 +/- 0.3 micro m(2) s(-1). The diffusion coefficient of exogenously added retinol was 3.2 +/- 0.5 micro m(2) s(-1). These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 +/- 0.2 micro m(2) s(-1). Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 +/- 0.01 micro m(2) s(-1), again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 +/- 0.01 micro m(2) s(-1). In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises approximately 1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all-trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all-trans retinol within the rod outer segment.     

Lipoperoxidation of rod outer segments of bovine retina is inhibited by soluble binding proteins for fatty acids

In the present study it was investigated if soluble-binding proteins for fatty acids (FABPs) present in neural retina show protection from in vitro lipoperoxidation of rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe⁺⁺ system, at 37°C during 90-120 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of soluble binding proteins for fatty acids. The fatty acid composition of rod outer segment membranes was substantially modified when subjected to non-enzymatic lipoperoxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3) and arachidonic acid (20:4 n-6). As a result of this, the unsaturation index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in the native and control membranes when compared with peroxidized ones. A similar decrease of chemiluminescence was observed with the addition of increasing concentrations of native or delipidated FABP retinal containing fractions to rod outer segment membranes. These results indicate that soluble proteins with fatty acid binding properties may act as antioxidant protecting rod outer segment membranes from deleterious effect.     

Oxidative damage of retinal rod outer segment membranes and the role of vitamin E

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N₂ atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin.We have found a large seasonal variation in the endogenous level of α-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of α-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol.This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.     

Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360 nm; emission, 457 or 540 nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30-60 min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65(-/-) mice, which lack 11-cis retinal.     

Characterization of the recombination reaction of rhodopsin.

The kinetics of recombination of 11-cis-retinal with bleached rod outer segments and sodium cholate solubilized rhodopsin have been investigated. At neutral pH, it was found that bleached rod outer segments in the presence of an excess of 11-cis-retinal follow pseudo-first-order kinetics. The results suggest the second-order formation of an intermediate addition compound followed by a first-order dehydration step to form a protonated aldimine linkage. In addition, at pH values above 7.5 or below 6.5 the kinetics of recombination are complex, indicating the formation of a molecular species inactive in recombination which is in equilibrium with the active form of opsin. Based upon the observed rate constants as a function of pH, a scheme is presented to describe the recombination reaction in bleached rod outer segments. The kinetics of recombination of sodium cholate solubilized opsin were also analyzed. In terms of formation of an intermediate addition compound and subsequent dehydration, the values for the individual rate constants for both bleached rod outer segments and cholate-solubilized opsin were found to compare very favorably. These results demonstrate that the sodium cholate (2 mg/ml) maintains opsin in a conformation very similar to that in the rod outer segment membrane and suggest that the cholate-opsin complex is an excellent model system for studies on opsin-membrane interactions.     

Differential phosphorylation by GTP and ATP in isolated rod outer segments of the retina.

Isolated bovine rod outer segment protein is phosphorylated with GTP-gamma-32P and ATP-gamma 32P and to a much lesser extent by CTP-gamma-32P and UTP-gamma-32P. Phosphorylation with both GTP (GTP-kinase activity) and ATP (ATP-kinase activity) is markedly stimulated by light; phosphorylation with GTP is lower in dark-adapted and higher in light-adapted rod outer segments than is phosphorylation with ATP. Km values of 20 and 200 muM and Vmax values of 2.1 and 5.9 nmol/(mg min(-1)) were calculated using ATP and GTP, respectively, in light-adapted outer segments. When outer segments are incubated with GTP-gamma-32P under the usual conditions employed in these experiments, no formation of ATP-gamma-32P was detected by the techniques of high-pressure liquid chromatography and thin-layer chromatography. In intact, light-bleached outer segments, GTP appears to specifically phosphorylate rhodopsin. Histone and phosvitin are not phosphorylated to any appreciable extent by GTP. Histone appears to block rhodopsin phosphorylation by GTP while histone and, to some extent, phosvitin, both act as substrates for ATP-kinase activity. Cyclic AMP and other adenine derivates have a marked inhibitory effect on GTP-kinase activity. Phosphate also inhibits GTP-kinase activity but stimulates ATP-kinase activity. Such differences in phosphorylation with GTP and ATP indicate that these activities are either due to separate enzyme systems or, if only one enzyme is involved, the activities are under separate physiological control in the photoreceptor unit.     

Interaction between photoexcited rhodopsin and peripheral enzymes in frog retinal rods. Influence on the postmetarhodopsin II decay and phosphorylation rate of rhodopsin

The major peripheral and soluble proteins in frog rod outer segment preparations, and their interactions with photoexcited rhodopsin, have been compared to those in cattle rod outer segments and found to be similar in both systems. In particular the GTP-binding protein (G) has the same subunit composition, the same abundance relative to rhodopsin (1/10) and it undergoes the same light and nucleotide-dependent interactions with rhodopsin in both preparations. Previous work on cattle rod outer segments has shown that photoexcited rhodopsin (R*), in a state identified with metarhodopsin II, associates with the G protein as a first step to the light-activated GDP/GTP exchange on G. The complex R*-G is stable in absence of GTP, but is rapidly dissociated by GTP owing to the GDP/GTP exchange reaction. Low bleaching extents (less than 10% R*) in absence of GTP therefore create predominantly R*-G complexes, whereas bleaching in presence of GTP creates free R*. We report here that, under conditions of complexed R*, two reactions of R* in frog rod outer segments are highly perturbed as compared to free R*: (a) the spectral decay of metarhodopsin II (MII) into later photoproducts, and (b) the phosphorylation of R* by an ATP-dependent protein kinase. a) The spectral measurements have been performed using linear dichroism on oriented frog rod outer segments; this technique allows discrimination between MII and later photoproducts absorbing at the same wavelength. Association of R* with G leads to a strong reduction of the amount of MIII formed and to an acceleration of the decay of MIII. Furthermore, MII is significantly stabilized, in agreement with the hypothesis that MII is the intermediate which binds to G. b) The phosphorylation of R* is strongly inhibited under conditions of R*-G complex formation as compared to free R*. Interferences between reactions at the three sites involved in R* are discussed: the retinal binding site in the hydrophobic core is sensitive to the presence of GTP-binding protein at its binding site on the cytoplasmic surface of R*; the kinase and the GTP-binding protein compete for access to their respective binding sites, both located on the surface of R*. We also observed a slow and nucleotide-dependent light-induced binding of a protein of molecular weight 50 000, which we consider as the equivalent of the 48 000 Mr light-dependent protein previously identified in cattle rod outer segments.     

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.     

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.     

Acylation of bovine rhodopsin by [3H]palmitic acid

Bovine retinas or preparations of rod outer segments incorporate [3H]palmitic acid into rhodopsin. The incorporation is both time- and temperature-dependent. The major product retains the chromatographic and electrophoretic properties of rhodopsin and remains photosensitive as demonstrated by alteration of its chromatographic behavior upon exposure to light. The incorporated radioactivity resists extraction with organic solvents and is not dissociated from the protein by detergents or under the denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive free fatty acid can, however, be released by alkaline hydrolysis. Hydroxylamine treatment yields a mixture of the free fatty acid and the fatty acyl hydroxamate. These results demonstrate the formation of an ester bond between [3H]palmitic acid and rhodopsin. Cycloheximide fails to inhibit the incorporation. This finding along with the ability of rod outer segments to support the incorporation point to the acylation of rhodopsin as a late post-translational event.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. I.

Guanosine 3′,5′-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concavanalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphosdiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.