Distribution of rhodopsin and retinochrome in the squid retina

The cephalopod retina contains two kinds of photopigments, rhodopsin and retinochrome. For many years retinochrome has been thought to be localized in the inner segments of the visual cells, whereas rhodopsin is in the outer segments. However, it is now clear that retinochrome can be extracted also from fragments of outer segments. In the dark-adapted retina of Loligo pealei retinochrome is distributed half-and-half in the inner and outer segments. Todarodes pacificus contains much more retinochrome than Loligo, and it is more abundant in the outer than in the inner segments. The outer segments of Loligo contain retinochrome and metarhodopsin in addition to rhodopsin, whether squids are kept in the dark or in the light. But there is extremely little metarhodopsin (about 3% of rhodopsin) even in light-adapted eyes. The inner segments contain only retinochrome, and much less in the light than in the dark. On the other hand, retinochrome in the outer segments increases markedly during light adaptation. These facts suggest the possibility that some retinochrome moves forward from the inner to the outer segments during light adaptation and there reacts with metarhodopsin to promote regeneration of rhodopsin.     

Histochemical localization of retinochrome and rhodopsin studied by fluorescence microscopy

Retinochrome is readily reduced by sodium borohydride into an N-retinyl protein that emits visible fluorescence upon irradiation with near-ultraviolet light. Rhodopsin is also converted to a similar fluorescent product, but only when denatured with formaldehyde before reduction. Based upon this difference, retinochrome was discriminated from rhodopsin on frozen sections. The distribution of these two photopigments in various photosensitive tissues was examined by means of epifluorescence microscopy. In the octopus retina (Octopus vulgaris), the yellow-green fluorescence of reduced retinochrome was observed in both the basal regions of the outer segments and throughout the inner segments of the visual cells, while the fluorescence of reduced rhodopsin was restricted to within the rhabdomal layer of the outer segments. In the squid parolfactory vesicles (Todarodes pacificus), rhodopsin was present in the central lumen, which contains the distal processes of the photoreceptor cells, while retinochrome was detected in the myeloid bodies scattered within the vesicular wall. In the slug retina (Limax flavus), rhodopsin was found in the microvilli, and retinochrome appeared to be concentrated in the photic vesicles of the visual cells.     

Retinochrome and rhodopsin in the extraocular photoreceptor of the squid, Todarodes

The deep-sea squid, Todarodes pacificus, possesses well-developed parolfactory vesicles as extraocular photoreceptors connected with the brain. The ventral set of vesicles forms a thread approximately 3mm long and looks orange owing to photopigments. The vesicle mainly consists of receptor cells, each of which is similar in structure to the visual cell, carrying rhabdomeres in the distal process and lamellated myeloid bodies in the proximal part. Recently we noticed that a crude extract of the vesicles is capable of isomerizing retinal from all-trans to the 11-cis form in the light, and confirmed that the vesicles in fact contained retinochrome in addition to rhodopsin. This is the first time that retinochrome has been detected in any place other than ocular tissues. The optical and chemical nature of these photopigments is the same as that we have observed in the Todarodes retina. Quantitative extractions have shown that the total yield of photopigments is approximately 0.0006 in absorbance at lambda max (light path, 10 mm) per milliliter per thread of vesicles, and that the amount of retinochrome in the vesicles is roughly equivalent to that of rhodopsin. Whereas rhodopsin is located in the rhabdomal membranes, retinochrome is probably associated with lamellated structures and their derivatives in the cytoplasm. In the parolfactory vesicles, retinochrome may also cooperate with rhodopsin in the same way as has been discussed for retinal photoreception.     

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.     

Kinetics of visual pigment regeneration in excised mouse eyes and in mice with a targeted disruption of the gene encoding interphotoreceptor retinoid-binding protein or arrestin.

Photoisomerization of 11-cis-retinal to all-trans-retinal and reduction to all-trans-retinol occur in photoreceptor outer segments whereas enzymatic esterification of all-trans-retinol, isomerization to 11-cis-retinol, and oxidation to 11-cis-retinal occur in adjacent cells. The processes are linked into a visual cycle by intercellular diffusion of retinoids. Knowledge of the mechanistic aspects of the visual cycle is very limited. In this study, we utilize chemical analysis of visual cycle retinoids to assess physiological roles for components inferred from in vitro experiments and to understand why excised mouse eyes fail to regenerate their bleached visual pigment. Flash illumination of excised mouse eyes or eyecups, in which regeneration of rhodopsin does not occur, produced a block in the visual cycle after all-trans-retinal formation; constant illumination of eyecups produced a block in the cycle after all-trans-retinol formation; and constant illumination of whole excised eyes resulted in a block of the cycle after formation of all-trans-retinyl ester. These blocks emphasize the role of cellular metabolism in the visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) has been postulated to play a role in intercellular retinoid transfer in the retina; however, the rates of recovery of 11-cis-retinal and of regeneration of rhodopsin in the dark in IRBP-/- mice were very similar to those found with wild-type (wt) mice. Thus, IRBP is necessary for photoreceptor survival but is not essential for a normal rate of visual pigment turnover. Arrestin forms a complex with activated rhodopsin, quenches its activity, and affects the release of all-trans-retinal in vitro. The rate of recovery of 11-cis-retinal in arrestin-/- mice was modestly delayed relative to wt, and the rate of rhodopsin recovery was approximately 80% of that observed with wt mice. Thus, the absence of arrestin appeared to have a minor effect on the kinetics of the visual cycle.     

FTIR studies of the photoactivation processes in squid retinochrome

Retinochrome is a photoisomerase of the invertebrate visual system, which converts all-trans-retinal to the 11-cis configuration and supplies it to visual rhodopsin. In this paper, we studied light-induced structural changes in squid retinochrome by means of low-temperature UV-visible and Fourier transform infrared (FTIR) spectroscopy. In PC liposomes, lumi-retinochrome was stable in the wide temperature range between 77 and 230 K. High thermal stability of the primary intermediate in retinochrome is in contrast to the case in rhodopsins. FTIR spectroscopy suggested that the chromophore of lumi-retinochrome is in a relaxed planar 11-cis form, being consistent with its high thermal stability. The chromophore binding pocket of retinochrome appears to accommodate both all-trans and 11-cis forms without a large distortion, and limited protein structural changes between all-trans and 11-cis chromophores may be suitable for the function of retinochrome as a photoisomerase. The analysis of N-D and O-D stretching vibrations in D(2)O revealed that the hydrogen bond of the Schiff base is weaker in retinochrome than in bovine rhodopsin and bacteriorhodopsin, while retinochrome has a water molecule under strongly hydrogen-bonded conditions (O-D stretch at 2334 cm(-)(1)). The hydrogen bond of the water is further strengthened in lumi-retinochrome. The formation of meta-retinochrome accompanies deprotonation of the Schiff base, together with the peptide backbone alterations of alpha-helices, and possible formation of beta-sheets. It was found that the Schiff base proton is not transferred to its counterion, Glu181, but directly released to the aqueous phase in PC liposomes (pH 7.5). This suggests that the Schiff base environment is exposed to solvent in meta-retinochrome, which may be advantageous for the hydrolysis reaction of the Schiff base in the transport of 11-cis-retinal to its shuttle protein.     

Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E) accumulate in retinal pigment epithelium in absence of light exposure: their origin is 11-cis-retinal

The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.     

Role of photoreceptor-specific retinol dehydrogenase in the retinoid cycle in vivo

The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH-/-) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH-/- mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.     

Ligand channeling within a G-protein-coupled receptor. The entry and exit of retinals in native opsin

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.     

Signaling states of rhodopsin. Formation of the storage form,metarhodopsin III,from active metarhodopsin II

Vertebrate rhodopsin consists of the apoprotein opsin and the chromophore 11-cis-retinal covalently linked via a protonated Schiff base. Upon photoisomerization of the chromophore to all-trans-retinal, the retinylidene linkage hydrolyzes, and all-trans-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-cis-retinal. All-trans-retinal release occurs in parallel with decay of the active form, metarhodopsin (Meta) II, in which the original Schiff base is intact but deprotonated. The intermediates formed during Meta II decay include Meta III, with the original Schiff base reprotonated, and Meta III-like pseudo-photoproducts. Using an intrinsic fluorescence assay, Fourier transform infrared spectroscopy, and UV-visible spectroscopy, we investigated Meta II decay in native rod disk membranes. Up to 40% of Meta III is formed without changes in the intrinsic Trp fluorescence and thus without all-trans-retinal release. NADPH, a cofactor for the reduction of all-trans-retinal to all-trans-retinol, does not accelerate Meta II decay nor does it change the amount of Meta III formed. However, Meta III can be photoconverted back to the Meta II signaling state. The data are described by two quasi-irreversible pathways, leading in parallel into Meta III or into release of all-trans-retinal. Therefore, Meta III could be a form of rhodopsin that is stored away, thus regulating photoreceptor regeneration.     

The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium

The recent identification of nonvisual opsins has revealed an expanding family of vertebrate opsin genes. The retinal pigment epithelium (RPE) and Müller cells contain a blue and UV light-absorbing opsin, the RPE retinal G protein-coupled receptor (RGR, or RGR opsin). The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore through a covalent Schiff base bond. In this study, the isomeric structure of the endogenous chromophore of RGR was identified by the hydroxylamine derivatization method. The retinaloximes derived from RGR in the dark consisted predominantly of the all-trans isomer. Irradiation of RGR with 470-nm monochromatic or near-UV light resulted in stereospecific isomerization of the bound all-trans-retinal to an 11-cis configuration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitro conditions, the photosensitivity of RGR is at least 34% that of bovine rhodopsin. These results provide evidence that RGR is bound in vivo primarily to all-trans-retinal and is capable of operating as a stereospecific photoisomerase that generates 11-cis-retinal in the pigment epithelium.     

Metaretinochrome in membranes as an effective donor of 11-cis retinal for the synthesis of squid rhodopsin

Aporetinochrome, which is a protein moiety of retinochrome without chromophore retinal, is found in the membrane containing retinochrome. All of the prosthetic retinal of retinochrome in membranes, which is all-trans retinal, is bound to the chromophoric site on the protein moiety, with protonated Schiff bases showing an absorption band with the maximum at 495 nm. On exposure to light, retinochrome is converted to metaretinochrome at room temperature. The prosthetic retinals of metaretinochrome in membranes, which are 11-cis retinals, are in two states: retinals bound to the chromophoric site with protonated Schiff bases, and the free retinals, which are separated from the protein moiety. These states are suggested from the following observations. (a) The ratio of the absorbance at 470 nm of metaretinochrome to that at 495 nm of the parental retinochrome differs because of differences in samples and is higher in the purer preparations. (b) The difference spectrum of absorption of metaretinochrome caused by alkalinization shows two minimum peaks at approximately 420 and 470 nm. (c) The rate of bleaching of metaretinochrome in membranes with dilute NH2OH is much faster than that of retinochrome, and the absorption band in the near- UV region is more susceptible to NH2OH than the visible absorption band. The state of the prosthetic retinals in metaretinochrome was confirmed directly by the reaction of metaretinochrome in membranes with NaBH4. After treatment with NaBH4, the sodium dodecyl sulfate- polyacrylamide gel electrophoretic pattern shows two fluorescent bands: one at the position that corresponds to the retinochrome protein (mol wt 27,000 +/- 2,000), and another at the front of migration, where no band of protein is observed. Retinoids extracted from the NaBH4-treated metaretinochrome in membranes and analyzed with high-pressure liquid chromatography show a main peak of 11-cis retinol. The results of this and earlier (Seki et al., 1982) papers are summarized, and it is strongly suggested that metaretinochrome in the squid retina may play the role of 11-cis retinal donor for opsin and contribute to the synthesis of the squid rhodopsin.     

The retinal G protein-coupled receptor (RGR) enhances isomerohydrolase activity independent of light

Rod and cone visual pigments use 11-cis-retinal, a vitamin A derivative, as their chromophore. Light isomerizes 11-cis- into all-trans-retinal, triggering a conformational transition of the opsin molecule that initiates phototransduction. After bleaching all-trans-retinal leaves the opsin, and light sensitivity must be restored by regeneration of 11-cis-retinal. Under bright light conditions the retinal G protein-coupled receptor (RGR) was reported to support this regeneration by acting as a photoisomerase in a proposed photic visual cycle. We analyzed the contribution of RGR to rhodopsin regeneration under different light regimes and show that regeneration, during light exposure and in darkness, is slowed about 3-fold in Rgr(-/-) mice. These findings are not in line with the proposed function of RGR as a photoisomerase. Instead, RGR, independent of light, accelerates the conversion of retinyl esters to 11-cis-retinal by positively modulating isomerohydrolase activity, a key step in the "classical" visual cycle. Furthermore, we find that light accelerates rhodopsin regeneration, independent of RGR.     

11-cis-retinal reduces constitutive opsin phosphorylation and improves quantum catch in retinoid-deficient mouse rod photoreceptors

Rpe65(-/-) mice produce minimal amounts of 11-cis-retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65(-/-) mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65(-/-) mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-cis-retinal to Rpe65(-/-) mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-cis-retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-cis-retinal, does not generate the activity generally associated with the bleached apoprotein.     

Docking of human rhodopsin mutant (Gly90→Asp) with beta-arrestin and cyanidin 3-rutinoside to cure night blindness

MOTIVATION: Rhodopsin is a visual pigment present in rod cells of retina. It belongs to GPCR family and involves photoisomerization of 11-cis-retinal to all-trans-retinal isomers, conformational changes in rhodopsin and signal transduction cascade to generate a nerve impulse. This signaling pathway has been targeted to eliminate the effect of a mutation (Gly90→Asp) responsible for abnormal activation of G-protein without retinal conformations in the absence of light leading to congenital night blindness. A theoretical model of rhodopsin with induced mutation has been deliberated in order to find potential ligands which can offset this mutational effect. The binding interactions between the target mutated rhodopsin model and potential ligands have been predicted with the help of molecular docking. The results indicated strong functional benefits of ligands as an inhibitor and an agonist for mutated rhodopsin model. Therefore, we propose a new visual cascade model which can initiate the normal signaling of rhodopsin mutant with the help of proposed ligands and can provide a hope for vision in future.     

HEK293S cells have functional retinoid processing machinery

Rhodopsin activation is measured by the early receptor current (ERC), a conformation-associated charge motion, in human embryonic kidney cells (HEK293S) expressing opsins. After rhodopsin bleaching in cells loaded with 11-cis-retinal, ERC signals recover in minutes and recurrently over a period of hours by simple dark adaptation, with no added chromophore. The purpose of this study is to investigate the source of ERC signal recovery in these cells. Giant HEK293S cells expressing normal wild-type (WT)-human rod opsin (HEK293S) were regenerated by solubilized 11-cis-retinal, all-trans-retinal, or Vitamin A in darkness. ERCs were elicited by flash photolysis and measured by whole-cell recording. Visible flashes initially elicit bimodal (R(1), R(2)) ERC signals in WT-HEK293S cells loaded with 11-cis-retinal for 40 min or overnight. In contrast, cells regenerated for 40 min with all-trans-retinal or Vitamin A had negative ERCs (R(1)-like) or none at all. After these were placed in the dark overnight, ERCs with outward R(2) signals were recorded the following day. This indicates conversion of loaded Vitamin A or all-trans-retinal into cis-retinaldehyde that regenerated ground-state pigment. 4-butylaniline, an inhibitor of the mammalian retinoid cycle, reversibly suppressed recovery of the outward R(2) component from Vitamin A and 11-cis-retinal-loaded cells. These physiological findings are evidence for the presence of intrinsic retinoid processing machinery in WT-HEK293S cells similar to what occurs in the mammalian eye.     

Signaling states of rhodopsin: absorption of light in active metarhodopsin II generates an all-trans-retinal bound inactive state

Absorption of light in rhodopsin leads through 11-cis- and all-trans-retinal isomerization, proton transfers, and structural changes to the active G-protein binding meta-II state. When meta-II is photolysed by blue light absorption, the activating pathway is apparently reverted, and rhodopsin is photoregenerated. However, the product formed, a P subspecies with A(max) = 500 nm (P(500)), is different from the ground state based on the following observations: (i) the ground state fingerprint of 11-cis-retinal does not appear in the infrared spectra, although the proton transfers and structural changes are reverted; (ii) extraction of the retinal from P(500) does not yield the expected stoichiometric amount of 11-cis-retinal but predominantly yields all-trans-retinal; (iii) the infrared spectrum of P(500) is similar to the classical meta-III intermediate, which arises from meta-II by thermal decay; and (iv) both P(500) and meta-III can be photoconverted to meta-II with the same changes in the infrared spectrum and without a significant change in the isomerization state of the extracted chromophore. The data indicate the presence of a "second switch" between active and inactive conformations that operates by photolysis but without isomerization around the C(11)-C(12) double bond. This emphasizes the exclusivity of the ground state, which is only accessible by the metabolic regeneration with 11-cis-retinal.     

Recovery of visual functions in a mouse model of Leber congenital amaurosis

The visual process is initiated by the photoisomerization of 11-cis-retinal to all-trans-retinal. For sustained vision the 11-cis-chromophore must be regenerated from all-trans-retinal. This requires RPE65, a dominant retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of all-trans-retinyl esters in the retinal pigment epithelium, lack of 11-cis-retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623-8628) that in Rpe65-/- mice, oral administration of 9-cis-retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9-cis-retinal administration significantly attenuated retinal ester accumulation and supported rod retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65-/- mice. This rod function is likely a consequence of low efficiency production of 11-cis-retinal by photo-conversion of all-trans-retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65-/- mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and retinal degenerations.     

Fourier transform infrared spectroscopic study on retinochrome and its primary photoproduct, lumiretinochrome

Structural studies of retinochrome, and its photoproduct, lumiretinochrome, were done by Fourier transform infrared difference spectroscopy. The absorption bands in the carbonyl stretching region which shift in D2O show the changes in the protein part during the photoreaction. Strong absorption bands in the finger-print region show that the all-trans-retinal chromophore in retinochrome isomerizes to the 11-cis-retinal chromophore in lumiretinochrome upon illumination with yellow-green light at 83K.     

Mole quantity of RPE65 and its productivity in the generation of 11-cis-retinal from retinyl esters in the living mouse eye

RPE65, a protein expressed in cells of the retinal pigment epithelium of the eye, is essential for the synthesis by isomerohydrolase of 11-cis-retinal, the chromophore of rod and cone opsins. Recent work has established that RPE65 is a retinyl ester binding protein, and as all-trans-retinyl esters are the substrate for isomerohydrolase activity, the hypothesis has emerged that RPE65 serves to deliver substrate to this enzyme or complex. We bred mice with five distinct combinations of the RPE65 Leu450/Met450 variants (Leu/Leu, Met/Met, Leu/Met, Leu/-, and Met/-), measured in mice of each genotype the mole quantity of RPE65 per eye, and measured the initial rate of rhodopsin regeneration after a nearly complete bleach of rhodopsin to estimate the maximum rate of 11-cis-retinal synthesis in vivo. The quantity of RPE65 per eye ranged from 5.7 pmol (Balb/c) to 0.32 pmol (C57BL/6N x Rpe65(-)(/)(-)); the initial rate of rhodopsin regeneration was a Michaelis function of RPE65, where V(max) = 18 pmol/min per eye and K(m) = 1.7 pmol, and not dependent on the Leu450/Met450 variant. At RPE65 levels well below the K(m), the rate of production of 11-cis-retinal per RPE65 molecule was approximately 10 min(-)(1). Thus, the results imply that as a chaperone each RPE65 molecule can deliver retinyl ester to the isomerohydrolase at a rate of 10 molecules/min; should RPE65 itself be identified as the isomerase, each copy must be able to produce at least 10 molecules of 11-cis-retinal per minute.