Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Retinaldehyde”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,477 records · Page 82Linked to original sources
PubMed2000

The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy.

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (K(m) approximately 75 micrometer V(max) approximately 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The K(m) for ATP decreased to approximately 25 micrometer after reconstitution, whereas the V(max) was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a V(max) for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.

ATP-Binding Cassette Transporters↗
PubMed2000

Signaling states of rhodopsin. Retinal provides a scaffold for activating proton transfer switches.

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.

Animals↗
PubMed2000

Bacteriorhodpsin experiences light-induced conformational alterations in nonisomerizable C(13)=C(14) pigments. A study with EPR.

The mechanism by which bacteriorhodopsin is activated following light absorption is not completely clear. We have detected protein conformational alterations following light absorption by retinal-based chromophores in the bacteriorhodopsin binding site by monitoring the rate of reduction-oxidation reactions of covalently attached spin labels, using EPR spectroscopy. It was found that the reduction reaction with hydroxylamine is light-catalyzed in the A103C-labeled pigment but not in E74C or M163C. The reaction is light-catalyzed even when isomerization of the C(13)=C(14) bond of the retinal chromophore is prevented. The reverse oxidation reaction with molecular oxygen is effective only in apomembrane derived from the mutant A103C. This reaction is light-accelerated following light absorption of the retinal oxime, which occupies the binding site. The light-induced acceleration is evident also in "locked" bacteriorhodopsin in which isomerization around the C(13)=C(14) bond is prevented. It is evident that the chromophore-protein covalent bond is not a prerequisite for protein response. In contrast to the case of the retinal oxime, a reduced C=N bond A103C-labeled pigment did not exhibit acceleration of the oxidation reaction following light absorption. Acceleration was observed, however, following substitution of the polyene by groups that modify the excited state charge delocalization. It is suggested that protein conformational alterations are induced by charge redistribution along the retinal polyene following light absorption.

Bacteriorhodopsins↗
PubMed2000

cDNA cloning and expression of a human aldehyde dehydrogenase (ALDH) active with 9-cis-retinal and identification of a rat ortholog, ALDH12.

This report describes the isolation of a heretofore uncharacterized aldehyde dehydrogenase (ALDH) with retinal dehydrogenase activity from rat kidney and the cloning and expression of a cDNA that encodes its human ortholog, the previously unknown ALDH12. The human ALDH12 cDNA predicts a 487-residue protein with the 23 invariant amino acids, four conserved regions, cofactor binding motif (G(209)XGX(3)G), and active site cysteine residue (Cys(287)) that typify members of the ALDH superfamily. ALDH12 seems at least as efficient (V(m)/K(m)) in converting 9-cis-retinal into the retinoid X receptor ligand 9-cis-retinoic acid as two previously identified ALDHs with 9-cis-retinal dehydrogenase activity, rat retinal dehydrogenase (RALDH) 1 and RALDH2. ALDH12, however, has approximately 40-fold higher activity with 9-cis- retinal than with all-trans-retinal, whereas RALDH1 and RALDH2 have equivalent and approximately 4-fold less efficiencies for 9-cis-retinal versus all-trans-retinal, respectively. Therefore, ALDH12 is the first known ALDH to show a preference for 9-cis-retinal relative to all-trans-retinal. Evidence consistent with the possibility that ALDH12 could function in a pathway of 9-cis-retinoic acid biosynthesis in vivo includes biosynthesis of 9-cis-retinoic acid from 9-cis-retinol in cells co-transfected with cDNAs encoding ALDH12 and the 9-cis-retinol/androgen dehydrogenase, cis-retinoid/androgen dehydrogenase type 1. Intense ALDH12 mRNA expression in adult and fetal liver and kidney, two organs that reportedly have relatively high concentrations of 9-cis-retinol, reinforces this notion.

Aldehyde Dehydrogenase↗
PubMed2000

A fully functional rod visual pigment in a blind mammal. A case for adaptive functional reorganization?

In the blind subterranean mole rat Spalax ehrenbergi superspecies complete ablation of the visual image-forming capability has been accompanied by an expansion of the bilateral projection from the retina to the suprachiasmatic nucleus. We have cloned the open reading frame of a visual pigment from Spalax that shows >90% homology with mammalian rod pigments. Baculovirus expression yields a membrane protein with all functional characteristics of a rod visual pigment (lambda(max) = 497 +/- 2 nm; pK(a) of meta I/meta II equilibrium = 6.5; rapid activation of transducin in the light). We not only provide evidence that this Spalax rod pigment is fully functional in vitro but also show that all requirements for a functional pigment are present in vivo. The physiological consequences of this unexpected finding are discussed. One attractive option is that during adaptation to a subterranean lifestyle, the visual system of this mammal has undergone mosaic reorganization, and the visual pigments have adapted to a function in circadian photoreception.

Amino Acid Sequence↗
PubMed2000

brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum.

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.

Bacteriorhodopsins↗
PubMed2001

ABCR, the ATP-binding cassette transporter responsible for Stargardt macular dystrophy, is an efficient target of all-trans-retinal-mediated photooxidative damage in vitro. Implications for retinal disease.

A large body of experimental and clinical data have documented the damaging effects of light exposure on photoreceptor cells although the identities of the biologically relevant molecular targets of photodamage are still uncertain. Several lines of evidence point to retinoids or retinoid derivatives as chromophores that can mediate light damage. We report here that ABCR, a photoreceptor-specific transporter involved in the recycling of all-trans-retinal, is unusually sensitive to photooxidation damage mediated by all-trans-retinal in vitro. Partial loss of ABCR function is responsible for Stargardt macular dystrophy, which is associated with accumulation of A2E, a diretinoid adduct within the retinal pigment epithelium. Photodamage to ABCR causes it to aggregate in SDS gels and results in the loss of retinal-stimulated ATPase activity. Peripherin/RDS and ROM-1, two structural proteins that colocalize with ABCR at the outer segment disc rim, are also significantly more susceptible to all-trans-retinal-mediated photodamage than are the major proteins from the rod outer segment. These observations imply that there may be specific protein targets of photodamage within the outer segment, and they may be especially relevant to assessing the risk of light exposure in those individuals who already have diminished ABCR activity due to mutation in one or both copies of the ABCR gene.

ATP-Binding Cassette Transporters↗
PubMed2001

Interaction of 11-cis-retinol dehydrogenase with the chromophore of retinal g protein-coupled receptor opsin.

Vertebrate opsins in both photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11-cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) of the RPE and Müller cells is an abundant opsin that generates 11-cis-retinal by stereospecific photoisomerization of its bound all-trans-retinal chromophore. We have analyzed a 32-kDa protein (p32) that co-purifies with bovine RGR from RPE microsomes. The co-purified p32 was identified by mass spectrometric analysis as 11-cis-retinol dehydrogenase (cRDH), and enzymatic assays have confirmed the isolation of an active cRDH. The co-purified cRDH showed marked substrate preference to 11-cis-retinal and preferred NADH rather than NADPH as the cofactor in reduction reactions. cRDH did not react with endogenous all-trans-retinal bound to RGR but reacted specifically with 11-cis-retinal that was generated by photoisomerization after irradiation of RGR. The reduction of 11-cis-retinal to 11-cis-retinol by cRDH enhanced the net photoisomerization of all-trans-retinal bound to RGR. These results indicate that cRDH is involved in the processing of 11-cis-retinal after irradiation of RGR opsin and suggest that cRDH has a novel role in the visual cycle.

Alcohol Oxidoreductases↗
PubMed2001

Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A.

In vertebrates, symmetric versus asymmetric cleavage of beta-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan beta,beta-carotene-15,15'-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving beta-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of beta-carotene at the 9',10' double bond of beta-carotene and resulting in the formation of beta-apo-10'-carotenal and beta-ionone, a substance known as a floral scent from roses, for example. Besides beta-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the beta,beta-carotene-15,15'-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

Amino Acid Sequence↗
PubMed2001

Biochemical defects in 11-cis-retinol dehydrogenase mutants associated with fundus albipunctatus.

Mutations in the gene encoding 11-cis-retinol dehydrogenase (RDH5; EC ) are associated with fundus albipunctatus, an autosomal recessive eye disease characterized by stationary night blindness and accumulation of white spots in the retina. In addition, some mutated alleles are associated with development of cone dystrophy, especially in elderly patients. The numbers of identified RDH5 mutations linked to fundus albipunctatus have increased considerably during recent years. In this work, we have characterized the biochemical and cell biological properties of 11 mutants of RDH5 to understand the molecular pathology of the disease. All RDH5 mutants showed decreased protein stability and subcellular mislocalization and, in most cases, loss of enzymatic activity in vitro and in vivo. Surprisingly, mutant A294P displays significant enzymatic activity. Cross-linking studies and molecular modeling showed that RDH5 is dimeric, and co-expression analyses of wild-type and mutated alleles showed that the mutated enzymes, in a trans-dominant-negative manner, influenced the in vivo enzymatic properties of functional variants of the enzyme, particularly the A294P mutant. Thus, under certain conditions, nonfunctional alleles act in a dominant-negative way on functional but relatively unstable mutated alleles. However, in heterozygous individuals carrying one wild-type allele, the disease is recessive, probably due to the stability of the wild-type enzyme.

Alcohol Oxidoreductases↗
PubMed2001

Synthesis of the all-trans-retinal chromophore of retinal G protein-coupled receptor opsin in cultured pigment epithelial cells.

Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cis isomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[(3)H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[(3)H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[(3)H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis.

Animals↗
PubMed2002

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.

Animals↗
PubMed2002

Recombinant class I aldehyde dehydrogenases specific for all-trans- or 9-cis-retinal.

The molecular basis for the specificity of aldehyde dehydrogenases (ALDHs) for retinal, the precursor of the morphogen retinoic acid, is still poorly understood. We have expressed in Escherichia coli both retinal dehydrogenase (RALDH), a cytosolic aldehyde dehydrogenase originally isolated from rat kidney, and the highly homologous phenobarbital-induced aldehyde dehydrogenase (PB-ALDH). Oxidation of propanal was observed with both enzymes. On the other hand, recombinant RALDH efficiently catalyzed oxidation of 9-cis- and all-trans-retinal, whereas PB-ALDH was inactive with all-trans-retinal and poorly active with 9-cis-retinal. A striking difference between PB-ALDH and all other class I ALDHs is the identity of the amino acid immediately preceding the active nucleophile Cys(302) (Ile(301) instead of Cys(301)). Nevertheless, these amino acids could be exchanged in either RALDH or PB-ALDH without affecting substrate specificity. Characterization of chimeric enzymes demonstrates that distinct groups of amino acids control the differential activity of RALDH and PB-ALDH with all-trans- and 9-cis-retinal. Of 52 divergent amino acids, the first 17 are crucial for activity with all-trans-retinal, whereas the next 25 are important for catalysis of 9-cis-retinal oxidation. Recombinant enzymes with specificity for all-trans- or 9-cis-retinal were obtained, which should provide useful tools to study the relative importance of local production of all-trans- versus 9-cis-retinoic acid in development and tissue differentiation.

Aldehyde Dehydrogenase↗
PubMed2002

Rhodopsin with 11-cis-locked chromophore is capable of forming an active state photoproduct.

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C(11)=C(12) double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C(11)=C(12) double bond is prevented by a 6-member ring structure (Rh(6.10)). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C(11)=C(12) double bond being locked, isomerization around the C(9)=C(10) or the C(13)=C(14) double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.

Hydrogen-Ion Concentration↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2002

Biochemical and physiological properties of rhodopsin regenerated with 11-cis-6-ring- and 7-ring-retinals.

Phototransduction is initiated by the photoisomerization of rhodopsin (Rho) chromophore 11-cis-retinylidene to all-trans-retinylidene. Here, using Rho regenerated with retinal analogs with different ring sizes, which prevent isomerization around the C(11)=C(12) double bond, the activation mechanism of this G-protein-coupled receptor was investigated. We demonstrate that 11-cis-7-ring-Rho does not activate G-protein in vivo and in vitro, and that it does not isomerize along other double bonds, suggesting that it fits tightly into the binding site of opsin. In contrast, bleaching 11-cis-6-ring-Rho modestly activates phototransduction in vivo and at low pH in vitro. These results reveal that partial activation is caused by isomerization along other double bonds in more rigid 6-locked retinal isomers and protonation of key residues by lowering pH in 11-cis-6-ring-Rhos. Full activation is not achieved, because isomerization does not induce a complete set of conformational rearrangements of Rho. These results with 6- and 7-ring-constrained retinoids provide new insights into Rho activation and suggest a potential use of locked retinals, particularly 11-cis-7-ring-retinal, to inactivate opsin in some retinal degeneration diseases.

Animals↗
PubMed2002

Dual-substrate specificity short chain retinol dehydrogenases from the vertebrate retina.

Retinoids are chromophores involved in vision, transcriptional regulation, and cellular differentiation. Members of the short chain alcohol dehydrogenase/reductase superfamily catalyze the transformation of retinol to retinal. Here, we describe the identification and properties of three enzymes from a novel subfamily of four retinol dehydrogenases (RDH11-14) that display dual-substrate specificity, uniquely metabolizing all-trans- and cis-retinols with C(15) pro-R specificity. RDH11-14 could be involved in the first step of all-trans- and 9-cis-retinoic acid production in many tissues. RDH11-14 fill the gap in our understanding of 11-cis-retinal and all-trans-retinal transformations in photoreceptor (RDH12) and retinal pigment epithelial cells (RDH11). The dual-substrate specificity of RDH11 explains the minor phenotype associated with mutations in 11-cis-retinol dehydrogenase (RDH5) causing fundus albipunctatus in humans and engineered mice lacking RDH5. Furthermore, photoreceptor RDH12 could be involved in the production of 11-cis-retinal from 11-cis-retinol during regeneration of the cone visual pigments. These newly identified enzymes add new elements to important retinoid metabolic pathways that have not been explained by previous genetic and biochemical studies.

Alcohol Oxidoreductases↗
PubMed2002

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.

Animals↗