Flash photolysis of rhodopsin in the cat retina

The bleaching of rhodopsin by short-duration flashes of a xenon discharge lamp was studied in vivo in the cat retina with the aid of a rapid, spectral-scan fundus reflectometer. Difference spectra recorded over a broad range of intensities showed that the bleaching efficacy of high-intensity flashes was less than that of longer duration, steady lights delivering the same amount of energy. Both the empirical results and those derived from a theoretical analysis of flash photolysis indicate that, under the conditions of these experiments, the upper limit of the flash bleaching of rhodopsin in cat is approximately 90%. Although the fact that a full bleach could not be attained is attributable to photoreversal, i.e., the photic regeneration of rhodopsin from its light-sensitive intermediates, the 90% limit is considerably higher than the 50% (or lower) value obtained under other experimental circumstances. Thus, it appears that the duration (approximately 1 ms) and spectral composition of the flash, coupled with the kinetic parameters of the thermal and photic reactions in the cat retina, reduce the light-induced regeneration of rhodopsin to approximately 10%.     

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.     

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.     

Blue light and the regeneration of human rhodopsin in situ

Hubbard has found that the photoisomerization of retinene was important for the regeneration of rhodopsin in vitro, and the object of the present investigation was to find whether this was also true for regeneration in the living human eye. In the Appendix is described a device which permits the rhodopsin density to be measured by analysing the light reflected from the fundus oculi in an ophthalmoscopic arrangement, the measurement taking about 5 seconds. Now if a blue and a yellow light viewed scotopically are adjusted in intensity so as to appear identical, they must bleach rhodopsin equally, but the blue will be more than 10 times as effective in isomerizing retinene. Therefore if retinene isomerization is important for rhodopsin regeneration, blue light should cause a more rapid regeneration after bleaching, and during bleaching the equilibrium level attained should be less profound. But, as the figures show, the course of bleaching and regeneration is identical for the matched yellow or blue bleaching lights, therefore isomerization of retinene is not important for rhodopsin regeneration in the living human eye.     

Neural and Photochemical Mechanisms of Visual Adaptation in the Rat

The effects of light adaptation on the increment threshold, rhodopsin content, and dark adaptation have been studied in the rat eye over a wide range of intensities. The electroretinogram threshold was used as a measure of eye sensitivity. With adapting intensities greater than 1.5 log units above the absolute ERG threshold, the increment threshold rises linearly with increasing adapting intensity. With 5 minutes of light adaptation, the rhodopsin content of the eye is not measurably reduced until the adapting intensity is greater than 5 log units above the ERG threshold. Dark adaptation is rapid (i.e., completed in 5 to 10 minutes) until the eye is adapted to lights strong enough to bleach a measurable fraction of the rhodopsin. After brighter light adaptations, dark adaptation consists of two parts, an initial rapid phase followed by a slow component. The extent of slow adaptation depends on the fraction of rhodopsin bleached. If all the rhodopsin in the eye is bleached, the slow fall of threshold extends over 5 log units and takes 2 to 3 hours to complete. The fall of ERG threshold during the slow phase of adaptation occurs in parallel with the regeneration of rhodopsin. The slow component of dark adaptation is related to the bleaching and resynthesis of rhodopsin; the fast component of adaptation is considered to be neural adaptation.     

"Rapid regeneration" in the cat retina: a case for spreading depression

Fundus reflectometry of the cat retina showed that under certain circumstances a rapid increase in density may follow intense bleaching exposures. The spectral characteristics of the density changes indicated that neither rhodopsin nor its bleach products could be responsible for this effect. The poor condition of the animals in which the phenomenon was observed and its conspicuous absence in the majority of the experimental runs suggested that the effect was associated with a process other than the resynthesis of rhodopsin. It was shown that an extrareceptoral event, spreading depression (SD) of the retina, is the most likely source of the rapid spectral change. The well-known tissue alterations associated with SD were induced in the retina independently of pigment density change. The resultant difference spectra resembled those produced when the rapid density increase occurred spontaneously. It seems likely that the abnormal physiological condition of those cats in which the phenomenon is more frequently observed primes the retina for the light-induced generation of spreading depression.     

Dark adaptation processes in the amphibian rod

Rod dark adaptation in the amphibian retina appears to be due to three processes: 1. background adaptation, occurring immediately after the extinction of an adapting or bleaching light, 2. intermediate adaptation, that frequently lasts 30 min or more and 3. opsin adaptation, which in the isolated retina where regeneration of rhodopsin is insignificant, is observed as a permanent loss of sensitivity after the completion of intermediate adaptation. Intermediate adaptation is characterized by a linear relation between log threshold and the amount of retinal present, a similar relation is obtained between log threshold and the amount of rhodopsin bleached in opsin adaptation.These adaptation processes are discussed in terms of a model of the rod outer segment.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Salt effects on the conformational stability of the visual G-protein-coupled receptor rhodopsin

Membrane protein stability is a key parameter with important physiological and practical implications. Inorganic salts affect protein stability, but the mechanisms of their interactions with membrane proteins are not completely understood. We have undertaken the study of a prototypical G-protein-coupled receptor, the α-helical membrane protein rhodopsin from vertebrate retina, and explored the effects of inorganic salts on the thermal decay properties of both its inactive and photoactivated states. Under high salt concentrations, rhodopsin significantly increased its activation enthalpy change for thermal bleaching, whereas acid denaturation affected the formation of a denatured loose-bundle state for both the active and inactive conformations. This behavior seems to correlate with changes in protonated Schiff-base hydrolysis. However, chromophore regeneration with the 11-cis-retinal chromophore and MetarhodopsinII decay kinetics were slower only in the presence of sodium chloride, suggesting that in this case, the underlying phenomenon may be linked to the activation of rhodopsin and the retinal release processes. Furthermore, the melting temperature, determined by means of circular dichroism and differential scanning calorimetry measurements, was increased in the presence of high salt concentrations. The observed effects on rhodopsin could indicate that salts favor electrostatic interactions in the retinal binding pocket and indirectly favor hydrophobic interactions at the membrane protein receptor core. These effects can be exploited in applications where the stability of membrane proteins in solution is highly desirable.     

The molar extinction of rhodopsin

The molar extinction of rhodopsin is 40,600 cm.² per mole equivalent of retinene; i.e., this is the extinction of a solution of rhodopsin which is produced by, or yields on bleaching, a molar solution of retinene. The molar extinctions of all-trans retinene and all-trans retinene oxime have also been determined in ethyl alcohol and aqueous digitonin solutions. On the assumption that each chromophoric group of rhodopsin is made from a single molecule of retinene, it is concluded that the primary photochemical conversion of rhodopsin to lumi-rhodopsin has a quantum efficiency of 1; though the over-all bleaching of rhodopsin in solution to retinene and opsin may have a quantum efficiency as low as one-half. On bleaching cattle rhodopsin, about two sulfhydryl groups appear for each molecule of retinene liberated. In frog rhodopsin the —SH:retinene ratio appears to be higher, 5:2 or perhaps even 3:1. Some of this sulfhydryl appears to have been engaged in binding retinene to opsin; some may have been exposed as the result of changes in opsin which accompany bleaching, comparable with protein denaturation.     

Dark regeneration of rhodopsin in crayfish photoreceptors

The eyes of crayfish were exposed to lights of known spectral composition, and the course of regeneration was followed in the dark by measuring the content of rhodopsin and metarhodopsin in single rhabdoms isolated at various times after the adaptation, using an assay that is based on the fluorescence of metarhodopsin. Complete recovery requires several days in the dark after intense adaptation to orange light, but requires less than 2 d after blue light exposure. Following an orange light exposure with blue produces recovery kinetics characteristic of the blue light exposure alone. This quickening of recovery occurs whether the receptors are exposed to blue light either immediately or many hours after the original exposure to orange. Conversely, following blue light adaptation with orange leads to slow recovery, which is characteristic of orange alone. Recovery from long-wavelength adaptation is slower principally because many rhabdoms seem to delay the onset of regeneration. We suggest that the regeneration system is itself photosensitive, and after orange light adaptation the supply of active chromophore (presumably 11-cis retinal) limits the rate of recovery. Once started, recovery proceeds slowly and continuously, and the total pigment concentration (rhodopsin plus metarhodopsin) in the rhabdomeric membrane remains approximately constant. Within hours after intense adapting exposures, the rhabdoms become altered in appearance, the surfaces become coated with accessory pigment, and the bands of microvilli are less distinct. These changes persist until recovery of rhodopsin proceeds, which suggests that visual pigment regeneration results from addition of newly synthesized rhodopsin associated with membrane turn-over.     

Immunoreactivity of rhodopsin and opsin

An examination by a radioimmunoassay of the relative affinity of opsin and rhodopsin for rabbit antibody raised against bovine rhodopsin revealed that opsin was the preferred antigen. About 10-fold greater amounts of rhodopsin than opsin were required to achieve 50% inhibition of binding of 125I-labeled ligand in the RIA. Opsin was more reactive when examined in the light or dark, compared to rhodopsin incubated in the dark. Mixtures of opsin and rhodopsin (prepared by partial bleaching of rhodopsin or synthetic mixtures) exhibited increased reactivity with increasing mole fraction of opsin. This response was nonlinear, with small increases in opsin producing relatively large increases in reactivity. A partial fractionation of the antibody into two groups showing differential reactivities toward opsin and rhodopsin was achieved by affinity chromatography on opsin-Sepharose. However, with both groups, opsin was still the preferred antigen. Scatchard analysis of 125I-labeled rhodopsin and opsin produced nonlinear plots, indicating the presence of multiple species of antibody. The affinities and binding capacities were similar for both labeled antigens. In competitive binding studies, the antibody showed a strong preference for either labeled ligand (rhodopsin or opsin) as compared to the unlabeled material. These latter observations indicate that altering rhodopsin either by bleaching or iodination produced changes in the relative immunoreactivity of the molecule.     

Effect of phospholipid removal on the kinetics of the metarhodopsin I to metarhodopsin II reaction.

The kinetics of the metarhodopsin (meta) I → metarhodopsin II reaction have been studied by flash photolysis in two different types of preparations of bovine rhodopsin: (i) digitonin-solubilized rod outer segment (ROS) membranes with a molar ratio of phospholipid to rhodopsin of approximately 90, and (ii) digitonin-solubilized phospholipid-free rhodopsin with a molar ratio of phospholipid to rhodopsin of less than 0.2. At 20 °C the kinetics in both preparations are multiexponential, but four terms are required to fit the data with the solubilized membranes, whereas only two are required with the phospholipid-free preparation. Thus, phospholipid removal simplifies the kinetics of the meta I → meta II reaction, but the resulting preparation still does not show first-order kinetics. The ratio of the time constants of these two components with detergent-solubilized phospholipid-free rhodopsin was nearly equal to the values found with ROS particles, rhodopsin-phospholipid recombinants and intact rabbit eyes. This suggests a common origin for these two components in all these preparations and appears to exclude heterogeneity in bound phospholipid as the basis of these two-component kinetics.     

Dark adaptation,sensitivity, and rhodopsin level in the eye of the lobster,Homarus

Summary Dark adaptation of living lobsters was measured by recording the ERG at several temperatures in the range 5–20 °C following adapting flashes that convert about 70% of the rhodopsin to metarhodopsin. Recovery of log threshold is rapid, and at 10–20° is nearly complete in 10 min. Only at 5 °C is dark adaptation significantly slowed. Comparison of dark adaptation with data on regeneration of pigment (Bruno et al., 1977) is consistent with the hypothesis that as rhodopsin concentration rises and falls, its only effect on sensitivity is to alter the probability of quantum catch. This interpretation is further bolstered by observations on winter lobsters that have a 70% deficiency of rhodopsin without the concomitant increase in metarhodopsin that accompanies light adaptation. No effect of metarhodopsin on sensitivity was detected. These experiments support the growing body of evidence indicating that the relationship between rhodopsin concentration and log threshold is fundamentally different in the rhabdomeric photoreceptors of invertebrates and the rods and cones of vertebrates.This work was supported by USPHS research grant EY 00222 to Yale University. S.N.B. was aided by NIH Postdoctoral Fellowship EY 52378, by funds made available through the Unidel Foundation, and by a grant from the University of Delaware Research Foundation.     

Methylation of the active-site lysine of rhodopsin

Purified bovine rhodopsin was reductively methylated with formaldehyde and pyridine/borane with the incorporation of approximately 20 methyl groups in the protein. Rhodopsin contains 10 non-active-site lysines, which account for the uptake of the 20 methyl groups. The permethylated rhodopsin thus formed is active toward bleaching, regeneration with 11-cis-retinal, and the activation of the GTPase (G protein) when photolyzed. The critical active-site lysine of permethylated rhodopsin can be liberated by photolysis. This lysine can be reductively methylated at 4 degrees C. Methylation under these conditions leads to the incorporations of approximately 1.5 methyl groups per opsin molecule using radioactive formaldehyde, with the ratio of epsilon-dimethyllysine:epsilon-monomethyllysine:lysine being approximately 5:4:1. The modified opsin(s) can regenerate with 11-cis-retinal to produce a mixture of active-site methylated and unmethylated rhodopsins having a lambda max = 512 nm. Using [14C]formaldehyde and [3H]retinal followed by reduction of the Schiff base, digestion, and chromatography showed that the active-site N-methyllysine was bound to the retinal. Treatment of the methylated opsin mixture (containing 1.5 active-site methyl groups) with o-phthalaldehyde/mercaptoethanol to functionalize the opsin bearing unreacted lysine, followed by regeneration with 11-cis-retinal and chromatographic separation, led to the preparation of the pure active-site epsilon-lysine monomethylated rhodopsin with a lambda max = 520 nm, significantly shifted bathochromically from rhodopsin or permethylated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visual pigment and photoreceptor sensitivity in the isolated skate retina

Photoreceptor potentials were recorded extracellularly from the aspartate-treated, isolated retina of the skate (Raja oscellata and R. erinacea), and the effects of externally applied retinal were studied both electrophysiologically and spectrophotometrically. In the absence of applied retinal, strong light adaptation leads to an irreversible depletion of rhodopsin and a sustained elevation of receptor threshold. For example, after the bleaching of 60% of the rhodopsin initially present in dark-adapted receptors, the threshold of the receptor response stabilizes at a level about 3 log units above the dark-adapted value. The application of 11-cis retinal to strongly light-adapted photoreceptors induces both a rapid, substantial lowering of receptor threshold and a shift of the entire intensity-response curve toward greater sensitivity. Exogenous 11-cis retinal also promotes the formation of rhodopsin in bleached photoreceptors with a time-course similar to that of the sensitization measured electrophysiologically. All-trans and 13-cis retinal, when applied to strongly light-adapted receptors, fail to promote either an increase in receptor sensitivity or the formation of significant amounts of light-sensitive pigment within the receptors. However, 9-cis retinal isin. These findings provide strong evidence that the regeneration of visual pigment in the photoreceptors directly regulates the process of photochemical dark adaptation.     

Zinc-induced decrease of the thermal stability and regeneration of rhodopsin

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn(2+) on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn(2+) concentrations (0-50 microm Zn(2+)). The thermal bleaching process is accelerated in the presence of Zn(2+) with k rate constants, at 55 degrees C, of 0.028 +/- 0.002 min(-1) (0 microm Zn(2+)) and 0.056 +/- 0.003 min(-1) (50 microm Zn(2+)), corresponding to t(12) values of 24.4 +/- 1.6 min and 11.8 +/- 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E(a) increase at 50 microm Zn(2+) for the process, with deltaG++ decrease and increase in deltaH++ and deltaS++ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn(2+) in normal and pathological retinal function.     

Light-induced reorganization of phospholipids in rod disc membranes

The transbilayer redistribution of spin-labeled phospholipid analogues (SL-PL) with choline, serine, and ethanolamine head groups (PC, PS, and PE, respectively) was studied on intact disc vesicles of bovine rod outer segment membranes in the dark and after illumination. Redistribution was measured by the extraction of spin-labeled lipid analogues from the outer leaflet of membrane using the bovine serum albumin back-exchange assay. In the dark, PS was distributed asymmetrically, favoring the outer leaflet, whereas PC and PE showed small if any asymmetry. Green illumination for 1 min caused lipid head group-specific reorganization of SL-PL. Extraction of SL-PS by bovine serum albumin showed a fast transient (<10 min) enhancement, which was further augmented by a peptide stabilizing the active metarhodopsin II conformation. The data suggest a direct release of 1 molecule of bound PS per rhodopsin into the outer leaflet and subsequent redistribution between the two leaflets. SL-PE and SL-PC showed more complex kinetics, in both cases consistent with a prolonged period of reduced extraction (2 phospholipids per rhodopsin in each case). The different phases of SL-PL reorganization after illumination may be related to the formation and decay of the active rhodopsin species and to their subsequent regeneration process.     

Visual pigment spectra of the comma butterfly,<Emphasis Type="Italic"> Polygonia c-album</Emphasis>, derived from in vivo epi-illumination microspectrophotometry

The visual pigments in the compound eye of the comma butterfly, Polygonia c-album, were investigated in a specially designed epi-illumination microspectrophotometer. Absorption changes due to photochemical conversions of the visual pigments, or due to light-independent visual pigment decay and regeneration, were studied by measuring the eye shine, i.e., the light reflected from the tapetum located in each ommatidium proximal to the visual pigment-bearing rhabdom. The obtained absorbance difference spectra demonstrated the dominant presence of a green visual pigment. The rhodopsin and its metarhodopsin have absorption peak wavelengths at 532 nm and 492 nm, respectively. The metarhodopsin is removed from the rhabdom with a time constant of 15 min and the rhodopsin is regenerated with a time constant of 59 min (room temperature). A UV rhodopsin with metarhodopsin absorbing maximally at 467 nm was revealed, and evidence for a blue rhodopsin was obtained indirectly.     

Illumination of bovine photoreceptor membranes causes phosphorylation of both bleached and unbleached rhodopsin molecules

Bovine rod outer segments were given a series of flashes, each bleaching from 0.1% to 0.4% of the rhodopsin present. 9-cis-Retinal was then added, regenerating the bleaching pigment to isorhodopsin. The phosphorylated pigment species having either four and five or six and eight phosphates were isolated by chromatofocusing. The amounts of rhodopsin and isorhodopsin present in the phosphorylated species were determined spectrally. The species with four and five phosphates per rhodopsin were approximately 50% rhodopsin-50% isorhodopsin. The more highly phosphorylated species were almost entirely isorhodopsin. Presumably, the phosphorylated rhodopsin was phosphorylated without having been bleached. At a 4% bleach level, approximately 0.5 rhodopsin was phosphorylated with four to five phosphates for each rhodopsin that was bleached and phosphorylated.     

Biochemical properties of 9-cis- and all-trans-retinoylopsins

The stoichiometry of the reaction between [14C]-9-cis-retinoyl fluoride, a close isostere of 9-cis-retinal, and bovine opsin and the biochemical and spectral properties of this new pigment were investigated. The stoichiometry of retinoid incorporation is approximately one in dodecyl maltoside, a detergent in which opsin is capable of regeneration with 11-cis-retinal. Interestingly, in Ammonyx LO, a detergent that does not permit rhodopsin regeneration, the stoichiometry of binding is still approximately one. By contrast, heat-denatured opsin does not irreversibly bind substantial [14C]retinoyl fluoride. This result strongly suggests that the nucleophilicity of the active site lysine is retained in Ammonyx LO but that further conformational changes in the protein, required to form rhodopsin, are not possible. These results are all consistent with an active site directed mechanism for the irreversible reaction of 9-cis-retinoyl fluoride with opsin probably at the active site lysine residue. The ultraviolet spectra of 9-cis-retinoylopsin and its all-trans congener show gamma max's at 373 and 380 nm, respectively, somewhat bathochromically shifted from their respective model N-butylretinamides which absorb at 347 and 351 nm. Photolysis of both 9-cis- and all-trans-retinoylopsins leads to the same photostationary state. This shows that, as expected, photoisomerization without bleaching occurs. The photolysis of either 9-cis- or all-trans-retinoylopsin in the presence of the G protein (transducin) does not lead to the activation of the latter. This is consistent with the notion that a protonated Schiff base is critical for the function of rhodopsin.