Retinaldehyde Research Articles

Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: Simultaneous measurements of phototactic and photophobic responses

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all- trans retinal restored both photoresponses in a carotenoid-deficient mutant strain of Chlamydomonas reinhardtii in a dose-dependent manner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All- trans retinal was more effective than 11- cis retinal to regenerate both photobehavioral responses. Analogs having locked 11- cis configurations and a phenyl ring in the side chain also induced photoresponses, although at concentrations more than two orders of magnitude higher than all- trans retinal. According to the present assay method, the responses were hardly detectable in cells incubated with retinal analogs in which the 13-ene was locked in either its trans or cis configuration. The results strongly suggest that the isomerization of the 13–14 double bond is important for photobehavioral signal transduction and that a single retinal-dependent photoreceptor controls both phototactic and photophobic responses.

Circadian photoreception in the retinally degenerate mouse (rd/rd)

We have examined the effects of light on circadian locomotor rhythms in retinally degenerate mice (C57BL/6J mice homozygous for the rd allele: rd/rd). The sensitivity of circadian photoreception in these mice was determined by varying the irradiance of a 15 min light pulse (515 nm) given at circadian time 16 and measuring the magnitude of the phase shift of the locomotor rhythm. Experiments were performed on animals 80 days of age. Despite the loss of visual photoreceptors in the rd/rd retina, animals showed circadian responses to light that were indistinguishable from mice with normal retinas (rd/+ and +/+). While no photoreceptor outersegments were identified in the retina of rd/rd animals (80-100 days of age), we did identify a small number of perikarya that were immunoreactive for cone opsins, and even fewer cells that contained rod opsin. Using HPLC, we demonstrated the presence and photoisomerization of the rhodopsin chromophore 11-cis retinaldehyde. The rd/rd retinas contained about 2% of 11-cis retinaldehyde found in +/+ retinas. We have yet to determine whether the opsin immunoreactive perikarya or some other unidentified cell type mediate circadian light detection in the rd/rd retina.

The role of retinal photoisomerase in the visual cycle of the honeybee.

The compound eye of the honeybee has previously been shown to contain a soluble retinal photoisomerase which, in vitro, is able to catalyze stereospecifically the photoconversion of all-trans retinal to 11-cis retinal. In this study we combine in vivo and in vitro techniques to demonstrate how the retinal photoisomerase is involved in the visual cycle, creating 11-cis retinal for the generation of visual pigment. Honeybees have approximately 2.5 pmol/eye of retinal associated with visual pigments, but larger amounts (4-12 pmol/eye) of both retinal and retinol bound to soluble proteins. When bees are dark adapted for 24 h or longer, greater than 80% of the endogenous retinal, mostly in the all-trans configuration, is associated with the retinal photoisomerase. On exposure to blue light the retinal is isomerized to 11-cis, which makes it available to an alcohol dehydrogenase. Most of it is then reduced to 11-cis retinol. The retinol is not esterified and remains associated with a soluble protein, serving as a reservoir of 11-cis retinoid available for renewal of visual pigment. Alternatively, 11-cis retinal can be transferred directly to opsin to regenerate rhodopsin, as shown by synthesis of rhodopsin in bleached frog rod outer segments. This retinaldehyde cycle from the honeybee is the third to be described. It appears very similar to the system in another group of arthropods, flies, and differs from the isomerization processes in vertebrates and cephalopod mollusks.

Retinol is essential for growth of activated human B cells.

When EBV-transformed human B cells are removed from conventional cell cultures, washed, and seeded at a low cell density in serum-free medium, their growth potential is greatly diminished. Fresh serum restores the growth of low density B cell cultures. We have traced this restorative effect to an essential factor present in the lipid fraction of serum and have identified it as all-trans retinol. The identification is based on the close similarities of the factor isolated from serum with authentic all-trans retinol as revealed by mass spectrometry, HPLC chromatography, and the ability to stimulate the growth of lymphoblastoid cells in the bioassay. Retinol is active at concentrations equal to its concentration in serum. Retinol is also a requirement for growth in suspension cultures at cell densities of 3 x 10(5)/ml. Cells removed at any time from such exponentially growing cultures and transferred to retinol-free medium cease to grow and consequently die, whereas in the continued presence of retinol, cell growth is unabated. All-trans retinal can substitute for retinol, but retinoic acid fails to stimulate the growth of lymphoblastoid cells at physiological concentrations. Normal human B lymphocytes also require retinol as a costimulator of proliferation after activation by anti-mu antibody or Staphylococcus aureus (Cowan strain) bacteria. In serum, retinol is bound to retinol-binding protein, which in turn forms a complex with prealbumin. Accordingly, we find that B cells respond to retinol bound to its physiological serum carrier, retinol-binding protein. In conclusion, human B cells are critically dependent for optimal growth in cell culture on an external supply of retinol.

Transduction noise induced by 4-hydroxy retinals in rod photoreceptors

New visual pigments were formed with 4-hydroxy retinals in isolated vertebrate rod photoreceptors by exposing bleached rods from the tiger salamander, Ambystoma tigrinum, to lipid vesicles containing the analogues. Formation of physiologically active pigment was demonstrated by the restoration of sensitivity and by a shift of approximately 50 nm in the peak of both the visual pigment absorptance spectrum and rod spectral sensitivity spectrum from approximately 520 to approximately 470 nm for 11-cis 4-hydroxy retinal. Membrane current recordings from the inner segments of isolated rods revealed excess fluctuations in membrane current after formation of the new pigment in bleached cells or after exposure of unbleached cells to flashes in the presence of the analogue. The excess current fluctuations are similar to the fluctuations elicited by steady light producing a few discrete responses per second, a rate approximately 100 times greater than the normal rate of spontaneous events in darkness. These results suggest that analogues of retinal can produce alterations in the frequency of production of discrete responses in darkness in rod photoreceptors.

11- cis retinal formation in the light catalyzed by a retinal-binding protein from the honeybee retina

A retinal-binding protein purified from honeybee retina was able to combine with various isomers of retinal and to catalyze their photoisomerization mostly to 11- cis retinal, the isomer necessary for rhodopsin regeneration. A model of the enzymatic reactions was developed which best fits the experimental curves. The resulting values for the kinetic constants confirm that the isomerization from all- trans retinal to 11- cis retinal is the favoured reaction. The results are discussed in terms of a possible physiological role of this protein which, similarly to retinochrome ofcephalopods, may be involved in visual pigment regeneration. Retinal-binding protein; Photoisomerase; Retinal-isomerase; Rhodopsin regeneration; Visual pigment renewal

Cell biology of retinal photoreceptors and pigment epithelium

Retinal photoreceptors (rods and cones) and retinal pigment epithelium (RPE) are highly-polarized cells that reside in close association within the eye. They carry out their interactions from a time early in embryologic life until the demise of the organism. RPE cells position their basal surfaces on an elaborate membrane (Bruch's membrane) which is intercalated between this epithelial monolayer and the photoreceptor blood supply. Their apical surfaces face the light-sensitive organelles (outer segments) of the photoreceptors. Interactions between photoreceptors and RPE take place at this interface.Mechanisms underlying photoreceptor phototransduction are now understood to a significant degree. When a photon is absorbed by a rod or cone photopigment, its chromophore, 11-cis retinaldehyde (11-cis retinal), isomerizes to the all trans form (t-retinol) and the protein undergoes a conformational change as well. The latter event activates a retina-specific GTPase (a G-protein called transducin) which, in turn, activates a retina-specific cGMP phosphodiesterase (cGMP-PDE). This cGMP-PDE then reduces the intracellular levels of cGMP which results in the closure of Na+/Ca++channels in the photoreceptor outer segment plasma membrane. The cell hyperpolarizes as a result and release of neurotransmitter at the synaptic terminal is diminished.

A comparison of some photoreceptor characteristics in the pineal and retina

Immunocytochemistry with a rod-specific antiserum was used to study the post-hatch development (2 days–300 days) of photoreceptor elements within the pineal of the Japanese quail. At all ages staining was restricted to limited numbers of pinealocytes scattered throughout the gland. An enzyme-linked immunosorbent assay (ELISA), with the same rod-specific antibody, was then used to obtain a quantitative measure of rod opsin in total eye and pineal extracts in both the developing retina and pineal. The opsin content of both tissues shows a marked increase during the first 30 days after hatch and then plateaued to 0.84±0.02 nmoles opsin in the eye and 2.20±0.11 pmoles opsin equivalents in the pineal. The increase in opsin in the retina may be associated with continued post-hatch development of the photoreceptors. We then attempted to demonstrate the presence of the rhodopsin chromophore within pineal and retinal extracts using HPLC analysis. In both retinal and pineal extracts, 11-cis retinaldehyde was identified and a light-induced shift from the 11-cis to the all-trans isomer was clearly shown. This analysis also allowed us to calculate the total content of 11-cis and all-trans retinaldehyde (derived from both rod and non-rod photoreceptors) of the eye and pineal (eye: 1.7±0.2 nmoles; pineal: 4.6±0.5 pmoles). In the quail eye, the total amount of retinaldehyde is more than twice the amount of rod-like opsin. This probably reflects the large contribution of cones in the quail retina; the cone pigments will contribute to the retinaldehyde content but are not recognized by the rodspecific antibodies. In the pineal, we also found more than double the concentration of retinaldehyde than we would have predicted from the amount of rod-like opsin. These results, coupled with our immunocytochemical findings, suggest that the quail pineal contains at least two classes of photoreceptor, some ‘rod-like’, others ‘non rod-like’.

Retinoids bound to interstitial retinol-binding protein during light and dark-adaptation

High-performance liquid chromatography was used to determine the types and amounts of retinoids bound to interstitial retinol-binding protein (IRBP) during light- and dark-adaptation in frogs. IRBP was separated from CRBP and CRA1BP by ion-exchange chromatography and quantitated by determining the amount of Serva Blue R dye bound to it in stained sodium dodecyl sulfate polyacrylamide gels. The amount of IRBP was not significantly different in light- and dark-adapted eyes (0.15 ± 0.05 nmol/ eye compared with 0.18 ± 0.08 nmol/ eye). In the dark-adapted state, RBP bound mainly 11- cis retinol and 11- cis retinal in quantities that summed to about 1 mol/mol RBP. After the onset of light-adaptation, all-trans retinol increased from its very low dark-adapted evel, peaked at 0.2 mol/mol IRBP and then declined to the dark-adapted level again. Concomitantly, the otal retinoid bound to IRBP fell, mainly because there was a drop in the amount of 11- cis retinal. During dark-adaptation, the amount of 11- cis retinal increased. No significant changes were seen in the amount of 11- cis retinol in light and darkness. These findings support the hypothesis that when rhodopsin is bleached IRBP transports all- trans retinol from the retina to the pigment epithelium and that it delivers 11- cis retinal to the rod outer segments for rhodopsin regeneration.

Analysis of a soluble lipid-protein complex carrying endogenous 11-cis retinaldehyde from bovine retinal pigment epithelium.

A soluble lipid-protein complex in bovine retinal pigment epithelium is shown to carry endogenous 11-cis retinaldehyde, in the extent of 15% of the total 11-cis retinaldehyde found in this tissue. The complex, analyzed with respect to its chemical composition, exhibits a lipid composition close resembling the lipid composition of the rod outer segment membrane; the SDS-PAGE evidences the presence of a number of protein bands, two of which of 34 and 27 kDa appear glycoproteins. Finally, the lipid-protein complex exhibits a discrete level of a Cathepsin D-like protease activity. From the above, the possibility is discussed that the soluble lipid-protein complex could represent some phagolysosomal inclusion occurring in the pigment epithelial cells upon rod outer segment phagocytosis.

Properties of retinal-oxidizing enzyme activity in rat kidney

An enzyme activity which converts retinal to retinoic acid was found in the cytosol rat kidney. The oxidation of retinal was pH-, temperature-, time- and protein-dependent. Under the assay conditions employed, the oxidase activity had an apparent K m of 125 μM toward all- trans retinal. n-Propylgallate, butylated hydroxytoluene and quinacrine inhibited the reaction. The inhibition caused by quinacrine can be partly reversed by FAD. p-Hydroxymercuribenzoate, a sulfhydryl cross-linking agent, was a potent inhibitor. 4′-(9-Acridinylamino)methanesulfon-anisidide, an inhibitor of aldehyde oxidase, inhibited the reaction by 77% at a concentration of 3 mM. All- trans retinal reversed the inhibition caused by acetaldehyde and 2-aminobenzaldehyde. Retinol inhibited the reaction, but retinoic acid did not. The specific activity of the enzyme was increased by vitamin A deficiency. These data indicate that retinal-oxidizing enzyme activity found in the kidney is a sulfhydryl flavoprotein and its activity is dependent on the vitamin A levels of the tissues.

Analysis of retinoids by direct exposure probe mass spectrometry

Recently, our laboratory has investigated the depletion of Vitamin A and its metabolites in experimental animals. High Pressure Liquid Chromatography (HPLC) was used to measure retinal oxidase activity by monitoring the conversion of retinaldehyde (RALD) to retinoic acid (ROIC). In order to obtain more information about these compounds, a Direct Exposure Probe mass spectrometric method was developed to confirm the presence of ROIC and RALD in HPLC peaks. A rapid negative ion chemical ionization (NICI) method using ammonia as reagent gas was developed to detect the presence of ROIC and RALD with picogram sensitivity. The ROIC and RALD peaks were collected from HPLC, extracted with hexane, evaporated under nitrogen and reconstituted in ethanol before placing onto a rhenium filament with current programmed from 0-1.3 A at 50 mA/sec. The instrument employed was a Finnigan 4510 operated in the NICI mode and scanned from m/z 100-650 with a source temperature of 80 C. Other parameters were electron energy 140 eV and filament current at 0.25 mA. Prominent ions were generated at m/z 284 and m/z 300 for RALD and ROIC which were subsequently monitored in the selected ion monitoring mode. In summary, we have developed a rapid retinoid identification method (2.5 minutes) which is more sensitive (pg vs ng) than HPLC and does not require elaborate sample preparation or derivitization. This method can be used as an important adjunct to HPLC enzyme studies.

Altered protein-chromophore interaction in dicyclohexylcarbodiimide-modified purple membrane sheets

Previous studies of N,N′-dicyclohexylcarbodiimide (DCCD)-modified bacteriorhodopsin (Kenthal, R. et al. (1985) Biochemistry 24, 4275–4279) used reaction conditions (detergent micelles) that are not optimal for subsequent physical studies. The present work describes new conditions for reaction of bacteriorhodopsin with DCCD in intact purple membrane sheets in the presence of 4.5% (v/v) diethylether and light. Like the detergent reaction system, the reaction is light induced, incorporates approximately 1 mol [P 1·1C]DCCD per mol bacteriorhodopsin, and results in a bleached chromophore. Peptide mapping indicates that the likely site of modification in intact membranes is identical to the site in the detergent reaction system: Asp 115. The retinal chromophore of DCCD-modified purple membrane has an absorbance maximum at 390 nm and very little induced circular dichroism. The retinal is easily extracted in hexane, yielding a 3:1 ratio of all- trans to 13- cis retinal. Borohydride reduces the retinal onto the protein within the 1–71 region of the amino acid sequence. These results suggest that Asp-115 is near the retinal binding cavity of bacteriorhodopsin. When DCCD reacts with Asp 115, retinal is displaced from its binding site.

The effect of pH on the interactions in mixed monolayers between phosphatidylserine and all- Trans retinal

The interactions in mixed monolayers between phosphatidylserine and all- trans retinal have been studied at the nitrogen/water interface at 21.0 ± 0.5°C. The monolayers were spread on a 1 × 10 −3 M phosphate buffer containing in addition 2.25 m M NaCl. Three pH values of the subphase were investigated, 5.6, 7.0, and 8.1. Two different phosphatidylserine samples were used. One was the highly unsaturated lipid component extracted from the disk membrane, the other being a commercial, much less unsaturated lipid sample (main components: stearic and oleic acids). The results show that the protonation state of the lipid films does not modify to any measurable extent the mixing pattern of PS with all- trans retinal. On the other hand, the difference in aliphatic composition between the two PS samples has small, but definite, bearing on the miscibility of these components at the interface. These results are consistent with the fact that hydrophobic interactions dominate the thermodynamics of mixing these constituents at the nitrogen/water interface.

Photoisomerization of all-trans-retinal in organic solvents and organized media

Abstract The photochemistry of all-trans -retinal (ATR) in n -hexane, ethanol, aqueous sodium dodecylsulphate, n -butyl stearate and phosphatidylcholine—H 2 O media under direct irradiation (λ ⩾ 380 nm) has been investigated. Photostationary state compositions of retinal isomers have been determined. Results are discussed in terms of the effects of solvent polarity on the relative position of the n,π* and π,π* states of the excited polyene, the π electron density and the spatial requirements vis-a-vis the solvent-induced barrier for isomerization of different double bonds. Characteristics of UV absorption bands of ATR in different media are also discussed.

Refolding of bacteriorhodopsin in lipid bilayers: A thermodynamically controlled two-stage process

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. 1. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. 2. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all- trans retinal. 3. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly α-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.

A survey of 3-dehydroretinal as a visual pigment chromophore in various species of crayfish and other freshwater crustaceans

3-Dehydroretinal (vitamin A2 aldehyde) was found in the eyes of three species among 10 species of freshwater crayfish examined. Since dark-adapted eyes contained the 11-cis form of 3-dehydroretinal, this compound must be the chromophore of the visual pigment. 3-Dehydroretinal always coexisted with retinal (vitamin A1 aldehyde), indicating the presence of a rhodopsin-porphyropsin visual pigment system.

The visual cycle operates via an isomerase acting on all-trans retinol in the pigment epithelium.

Thirty years have elapsed since Wald and his colleagues showed that 11-cis retinal was isomerized to all-trans when rhodopsin was bleached, yet little has been understood about the reverse process that generates 11-cis retinal for rhodopsin regeneration. It is not known whether the isomerization is enzyme-mediated, whether it occurs in the pigment epithelium or in the retina, or whether retinal, retinol, or a retinyl ester is the vitamin A compound that is isomerized. Radiolabeled all-trans retinol and high-performance liquid chromatography have now been used to demonstrate the existence of an eye-specific, membrane-bound enzyme (retinol isomerase) that converts all-trans to 11-cis retinol in the dark. Retinol isomerase is concentrated in the pigment epithelium; this localization clarifies the role of this tissue in rhodopsin regeneration and explains the need to transfer all-trans retinol from the rod outer segments to the pigment epithelium during the visual cycle.

Binding of 11-cis retinaldehyde to the partially purified cellular retinaldehyde binding protein from bovine retinal pigment epithelium.

11-cis retinaldehyde binding analysis was performed on a bovine retinal pigment epithelium preparation of cellular retinaldehyde binding protein (CRALBP), whose purity degree was estimated as 75%. Equilibrium binding studies were carried out measuring the replacement of tritium-labeled with unlabeled 11-cis retinaldehyde at 25 degrees C. Analysis of the experimental data both by a direct curve-fitting procedure utilizing a non linear least square regression analysis and by a conventional Scatchard plot revealed a single non-interacting binding site with an apparent equilibrium constant of 0.9 X 10(-7) M. A binding stoichiometry of approximately 1 mol of 11-cis retinaldehyde/mol of binding protein can be calculated from the experimental data. Competition studies carried out in the presence of unlabeled 'trans' and 'cis' isomers of vitamin A derivatives confirm the high degree of specificity of the 11-cis retinaldehyde binding.

A study of visual pigments in the two antarctic crustaceans Orchomene plebs (Amphipoda) and Glyptonotus antarcticus (Isopoda)

Abstract 1. 1. Total chromophore contents as well as the contributions made by 11- cis retinal were determined by high pressure liquid chromatography in light- and dark-adapted eyes of Orchomene plebs and Glyptonotus antarcticus (Amphipoda and Isopoda, respectively). 2. 2. In O. plebs the highest amount of total chromophore in pmol/eye was found to be 18.5 in 36 hr dark-adapted animals. The lowest amount (11.6 pmol/eye) was recorded in 24 hr light-adapted individuals. 3. 3. In dark-adapted O. plebs , irrespective of whether dark-adapted for 36 or 60 hr, the percentage of 11- cis retinal was maximally 96.6%. In the light-adapted material it reached 71.2% 4. 4. In eyes of 20 hr dark-adapted Glyptonotous antarcticus , possibly because of insufficient dark adaptation, a total chromophore content of only 3.2 pmol/eye was found. The percentage of 11- cis retinal was 55.8. 5. 5. Porphyropsin with its testable 3-dehydroretinal (vitamin A 2 -aldehyde) was not encountered in any of our samples. 6. 6. Calculations of photopigment per gram body weight and a comparison with data from freshwater crayfish show that dark-adapted O. plebs possess approximately 20 times the relative photopigment amount of the crayfish. Absolute sensitivity of the eye of O. plebs is, therefore, expected to be very high.