CoA- and non-CoA-dependent retinol esterification in retinal pigment epithelium

Washed, buffered microsomes from bovine retinal pigment epithelium catalyze retinyl ester synthesis from retinol in the absence of an exogenous acyl donor. A plot of retinyl ester synthesis versus time reaches a plateau at 123 +/- 26 nmol of retinyl ester mg-1 microsomal protein, providing a minimum value of the concentration of the endogenous acyl donor. Fatty acyl-CoA analysis by three different methods employing high performance liquid chromatography resulted in the detection of less than 1 nmol mg-1 protein of acyl-CoA, indicating that fatty acyl-CoA is not the endogenous acyl donor. Stimulation of the rate of retinyl ester synthesis by palmitoyl-CoA or ATP, CoA, and palmitate is observed following its addition at the beginning of the reaction or after the endogenous acyl source has been exhausted by 20 min of reaction with retinol. Palmitate from [14C]palmitoyl-CoA is incorporated into retinyl ester at a rate similar to that for the incorporation of [3H] retinol, demonstrating the presence of an apparent acyl-CoA:retinol acyl transferase activity. The acyl group from palmitoyl-CoA can be transferred initially to a component of the microsomes and subsequently to retinol. The product of retinyl ester synthesis from all-trans-retinol and palmitoyl-CoA is all-trans-retinyl palmitate, indicating that the stereochemical configuration is retained during esterification. The kinetic parameters for the esterification of 11-cis-retinol and all-trans-retinol are similar.     

Chicken retinas contain a retinoid isomerase activity that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol

Vertebrate retinas contain two types of light-detecting cells. Rods subserve vision in dim light, while cones provide color vision in bright light. Both contain light-sensitive proteins called opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde by absorption of a photon. Restoration of light sensitivity requires chemical re-isomerization of retinaldehyde by an enzymatic pathway called the visual cycle in the retinal pigment epithelium. The isomerase in this pathway uses all-trans-retinyl esters synthesized by lecithin retinol acyl transferase (LRAT) as the substrate. Several lines of evidence suggest that cone opsins regenerate by a different mechanism. Here we demonstrate the existence of two catalytic activities in chicken retinas. The first is an isomerase activity that effects interconversion of all-trans-retinol and 11-cis-retinol. The second is an ester synthase that effects palmitoyl coenzyme A-dependent synthesis of all-trans- and 11-cis-retinyl esters. Kinetic analysis of these two activities suggests that they act in concert to drive the formation of 11-cis-retinoids in chicken retinas. These activities may be part of a new visual cycle for the regeneration of chromophores in cones.     

Inhibitors of retinyl ester formation also prevent the biosynthesis of 11-cis-retinol

Lecithin retinol acyl transferase (LRAT) from the retinyl pigment epithelium is potently inhibited by all-trans-retinyl alpha-bromoacetate in the micromolar range. The inhibition is competitive and reversible. The retinyl pigment epithelium also contains an enzymatic activity capable of converting added all-trans-retinol into 11-cis-retinol. This isomerization is likely to require the intermediate formation of all-trans-retinyl esters, which are themselves produced by LRAT action. Here this possibility is directly tested by studying the effect of all-trans-retinyl alpha-bromoacetate on the isomerization reaction. When pigment epithelium membranes are preincubated with all-trans-retinyl alpha-bromoacetate, they form neither retinyl esters nor 11-cis-retinol from added all-trans-retinol. However, if the pigment epithelium membranes are first allowed to form all-trans-retinyl esters from all-trans-retinol before the addition of all-trans-retinyl alpha-bromoacetate, then 11-cis-retinol formation proceeds at close to the rate found in the absence of inhibitor. In addition, 11-cis-retinyl esters are not formed under these conditions, eliminating the possibility of a direct ester-ester isomerization route. Therefore, all-trans-retinyl esters are obligate intermediates in the biosynthesis of 11-cis-retinol.     

Retinoid dynamics in chicken eye during pre-and postnatal development

Changes in the steady state level of retinols, retinaldehydes and retinyl esters in the trans and 11-cis forms and trans retinoic acid were measured in whole chicken eye during development from day 6in ovo to day 3 post-hatch. These retinoids, quantified by different HPLC systems, were detected in this time sequence: trans-retinol and trans-retinyl esters in the first weekin ovo, 11-cis-retinol in the second week. The highest level of 11-cis-retinaldehyde and 11-cis-retinyl esters was reached at the end of developmentin ovo; however, their levels increased further after hatching. The retinoic acid level decreased at the end of the first week, rising again at the end of the second week.The enzyme activities involved in the metabolism of these retinoids-acyl-CoA: retinol acyltransferase, trans-retinol dehydrogenase, 11-cis-retinol dehydrogenase, trans-retinyl ester hydrolase and trans: 11-cis-retinol isomerase were also estimated and they were detectable already in the first week of developmentin ovo.At day 6 of the biosynthesis of retinoic acid by the retinaldehyde dehydrogenase activity from retina cytosol was also shown.     

All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle

The identification of the critical enzyme(s) that carries out the trans to cis isomerization producing 11-cis-retinol during the operation of the visual cycle remains elusive. Confusion exists in the literature as to the exact nature of the isomerization substrate. At issue is whether it is an all-trans-retinyl ester or all-trans-retinol (vitamin A). As both putative substrates interconvert rapidly in retinal pigment epithelial membranes, the choice of substrate can be ambiguous. The two enzymes that effect interconversion of all-trans-retinol and all-trans-retinyl esters are lecithin retinol acyl transferase (LRAT) and retinyl ester hydrolase (REH). The retinyl ester or all-trans-retinol pools are radioactively labeled separately in the presence of inhibitors of LRAT and REH, effectively preventing their interconversion. Pulse-chase experiments unambiguously demonstrate that all-trans-retinyl esters, and not all-trans-retinol, are the precursors of 11-cis-retinol. When the all-trans-retinyl ester pool is radioactively labeled, the resulting 11-cis-retinol is labeled with the same specific activity as the precursor ester. The converse is true with vitamin A. These data unambiguously establish all-trans-retinyl esters as the precursors of 11-cis-retinol.     

Molecular logic of 11-cis-retinoid biosynthesis in a cone-dominated species

The biochemical pathway to visual chromophore biosynthesis in rod-dominated animals involves minimally a two component system in which all-trans-retinyl esters, generated by the action of lecithin retinol acyltransferase (LRAT) on vitamin A, are processed into 11-cis-retinol by isomerohydrolase. Possible differences in retinoid metabolism in cone-dominated animals have been noted in the literature, so it was of interest to explore whether these differences are tangential or fundamental. Central to this issue is whether cone-dominated animals use an isomerohydrolase (IMH)-based mechanism in the predominant pathway to 11-cis-retinoids. Here, it is shown that all-trans-retinyl esters (tREs) are the direct precursors of 11-cis-retinol formation in chicken retinyl pigment epithelium/retina preparations. This conclusion is based on at least three avenues of evidence. First, reagents that block tRE synthesis from vitamin A also block 11-cis-retinol synthesis. Second, pulse-chase experiments also establish that tREs are the precursors to 11-cis-retinol. Finally, 11-cis-retinyl-bromoacetate, a known affinity-labeling agent of isomerohydrolase, also blocks chromophore biosynthesis in the cone system.     

Aberrant metabolites in mouse models of congenital blinding diseases: formation and storage of retinyl esters

Regeneration of the visual chromophore, 11-cis-retinal, is a critical step in restoring photoreceptors to their dark-adapted conditions. This regeneration process, called the retinoid cycle, takes place in the photoreceptor outer segments and the retinal pigment epithelium (RPE). Disabling mutations in nearly all of the retinoid cycle genes are linked to human conditions that cause congenital or progressive defects in vision. Several mouse models with disrupted genes related to this cycle contain abnormal fatty acid retinyl ester levels in the RPE. To investigate the mechanisms of retinyl ester accumulation, we generated single or double knockout mice lacking retinoid cycle genes. All-trans-retinyl esters accumulated in mice lacking RPE65, but they are reduced in double knockout mice also lacking opsin, suggesting a connection between visual pigment regeneration and the retinoid cycle. Only Rdh5-deficient mice accumulate cis-retinyl esters, regardless of the simultaneous disruption of RPE65, opsin, and prRDH. 13-cis-Retinoids are produced at higher levels when the flow of retinoid through the cycle was increased, and these esters are stored in specific structures called retinosomes. Most importantly, retinylamine, a specific and effective inhibitor of the 11-cis-retinol formation, also inhibits the production of 13-cis-retinyl esters. The data presented here support the idea that 13-cis-retinyl esters are formed through an aberrant enzymatic isomerization process.     

Disruption of the 11-cis-retinol dehydrogenase gene leads to accumulation of cis-retinols and cis-retinyl esters

To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol dehydrogenase knockout mice, the appearance of the fundus was normal and punctata typical of this human hereditary ocular disease were not present. A second typical symptom associated with this disease is delayed dark adaptation. Homozygous 11-cis-retinol dehydrogenase mutants showed normal rod and cone responses. 11-cis-Retinol dehydrogenase knockout mice were capable of dark adaptation. At bleaching levels under which patients suffering from fundus albipunctatus could be detected unequivocally, 11-cis-retinol dehydrogenase knockout animals displayed normal dark adaptation kinetics. However, at high bleaching levels, delayed dark adaptation in 11-cis-retinol dehydrogenase knockout mice was noticed. Reduced 11-cis-retinol oxidation capacity resulted in 11-cis-retinol/13-cis-retinol and 11-cis-retinyl/13-cis-retinyl ester accumulation. Compared with wild-type mice, a large increase in the 11-cis-retinyl ester concentration was noticed in 11-cis-retinol dehydrogenase knockout mice. In the murine retinal pigment epithelium, there has to be an additional mechanism for the biosynthesis of 11-cis-retinal which partially compensates for the loss of the 11-cis-retinol dehydrogenase activity. 11-cis-Retinyl ester formation is an important part of this adaptation process. Functional consequences of the loss of 11-cis-retinol dehydrogenase activity illustrate important differences in the compensation mechanisms between mice and humans. We furthermore demonstrate that upon 11-cis-retinol accumulation, the 13-cis-retinol concentration also increases. This retinoid is inapplicable to the visual processes, and we therefore speculate that it could be an important catabolic metabolite and its biosynthesis could be part of a process involved in regulating 11-cis-retinol concentrations within the retinal pigment epithelium of 11-cis-retinol dehydrogenase knockout mice.     

Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein

In photoreceptor cells of the retina, photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction. Regeneration of 11-cis-retinal proceeds via a complex set of reactions in photoreceptors and in adjacent retinal pigment epithelial cells where all-trans-retinol is isomerized to 11-cis-retinol. Our results show that isomerization in vitro only occurs in the presence of apo-cellular retinaldehyde-binding protein. This retinoid-binding protein may drive the reaction by mass action, overcoming the thermodynamically unfavorable isomerization. Furthermore, this 11-cis-retinol/11-cis-retinal-specific binding protein potently stimulates hydrolysis of endogenous 11-cis-retinyl esters but has no effect on hydrolysis of all-trans-retinyl esters. Apo-cellular retinaldehyde-binding protein probably exerts its effect by trapping the 11-cis-retinol product. When retinoid-depleted retinal pigment epithelial microsomes were preincubated with different amounts of all-trans-retinol to form all-trans-retinyl esters and then [3H]all-trans-retinol was added, as predicted, the specific radioactivity of [3H]all-trans-retinyl esters increased during subsequent reaction. However, the specific radioactivity of newly formed 11-cis-retinol stayed constant during the course of the reaction, and it was largely unaffected by expansion of the all-trans-retinyl ester pool during the preincubation. The absence of dilution establishes that most of the ester pool does not participate in isomerization, which in turn suggests that a retinoid intermediate other than all-trans-retinyl ester is on the isomerization reaction pathway.     

Biochemical characterization of the retinoid isomerase system of the eye

We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.     

Hydrolysis of 11-cis- and all-trans-retinyl palmitate by homogenates of human retinal epithelial cells

The retinal epithelium plays an important role in the storage and metabolism of retinoids in the eye. Studies were conducted to examine the enzymatic hydrolysis of retinyl esters by human retinal epithelial cells. Homogenates prepared from these cells were found to hydrolyze both the 11-cis- and all-trans-isomers of retinyl palmitate. Retinyl ester hydrolysis was time-, protein-, and pH-dependent. The 11-cis isomer was hydrolyzed at a rate which was approximately 20 times greater than that of the all-trans isomer. The 11-cis-retinyl palmitate hydrolase activity did not require detergents, unlike the all-trans-retinyl palmitate hydrolase activity, which required detergents for activity. The 11-cis-retinyl palmitate hydrolase activity was maximally active with the addition of 1.0% sodium taurocholate at about pH 8.5, was abolished by incubation at 50 degrees C for 10 min, and was quantitatively recovered in the pellet after centrifugation at 100,000 X g for 1 h. The rate of hydrolysis of 11-cis-retinyl palmitate became saturated with increasing concentrations of 11-cis-retinyl palmitate; under the assay conditions employed, the hydrolase activity had an apparent Km of 19 microM toward 11-cis-retinyl palmitate. All-trans-retinol and 11-cis-retinyl did not affect the rate of hydrolysis of 11-cis-retinyl palmitate, and addition of all-trans-retinyl palmitate only weakly inhibited the 11-cis-retinyl palmitate hydrolytic activities. These data indicate that the human retinal epithelium possesses distinct activities for the hydrolysis of 11-cis- and all-trans-retinyl esters and raise the possibility that these activities may provide a means of distinguishing the stereoisomers of retinol in this tissue.     

Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation

Mutations in the human CRALBP gene cause retinal pathology and delayed dark adaptation. Biochemical studies have not identified the primary physiological function of CRALBP. To resolve this, we generated and characterized mice with a non-functional CRALBP gene (Rlbp1(-/-) mice). The photosensitivity of Rlbp1(-/-) mice is normal but rhodopsin regeneration, 11-cis-retinal production, and dark adaptation after illumination are delayed by >10-fold. All-trans-retinyl esters accumulate during the delay indicating that isomerization of all-trans- to 11-cis-retinol is impaired. No evidence of photoreceptor degeneration was observed in animals raised in cyclic light/dark conditions for up to 1 year. Albino Rlbp(-/-) mice are protected from light damage relative to the wild type. These findings support a role for CRALBP as an acceptor of 11-cis-retinol in the isomerization reaction of the visual cycle.     

Hydrolysis of 11-cis- and all-trans-retinyl palmitate by retinal pigment epithelium microsomes.

A partial characterization of the enzymatic hydrolysis of 11-cis- and all-trans-retinyl palmitate by bovine retinal pigment epithelium microsomes was carried out using a micro-radiometric method to quantitate liberated palmitic acid. Retinyl ester hydrolase (REH) activity was examined in the absence of detergent. Hydrolysis of 11-cis- and all-trans-retinyl palmitate was protein- and time-dependent. Optimal enzyme activity occurred at slightly alkaline pH (8-9). Apparent kinetic constants (Vmax and Km) for the 11-cis-REH were 2.1 nmol/min/mg protein and 66 microM, respectively. All-trans-REH demonstrated a lower maximum velocity of 0.3 nmol/min/mg protein and a slightly higher substrate affinity of 27 microM. Further characterization of 11-cis-retinyl palmitate hydrolysis involved monitoring formation of reaction products, 11-cis retinol and palmitic acid, which were found to be released in essentially a 1:1 stoichiometry. Addition of all-trans retinyl bromoacetate, a known inhibitor of lecithin:retinol acyltransferase reduced both 11-cis and all-trans-REH activities but to significantly different degrees (50 and 76%, respectively). Although the microsomal preparation exhibited LRAT activity, acyl transfer was not readily reversible as labeled palmitic acid was not transferred to added acyl acceptor compounds. These findings suggest that hydrolysis of 11-cis-retinyl palmitate by bovine retinal pigment epithelium microsomes may occur at a catalytic site distinct from that for the all-trans isomer and that this hydrolysis is not representative of a reverse transesterification reaction.     

RPE65 operates in the vertebrate visual cycle by stereospecifically binding all-trans-retinyl esters

RPE65 is a major protein of unknown function found associated with the retinyl pigment epithelial (RPE) membranes [Hamel, C. P., Tsilou, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Biol. Chem. 268, 15751-15757; Bavik, C. O., Levy, F., Hellman, U., Wernstedt, C., and Eriksson, U. (1993) J. Biol. Chem. 268, 20540-20546]. RPE65 knockouts fail to synthesize 11-cis-retinal, the chromophore of rhodopsin, and accumulate all-trans-retinyl esters in the RPE. Previous studies have also shown that RPE65 is specifically labeled with all-trans-retinyl ester based affinity labeling agents, suggesting a retinyl ester binding role for the protein. In the present work, we show that purified RPE65 binds all-trans-retinyl palmitate (tRP) with a K(D) = 20 pM. These quantitative experiments are performed by measuring the quenching of RPE65 fluorescence by added tRP. The binding for tRP is highly specific because 11-cis-retinyl palmitate binds with a K(D) = 14 nM, 11-cis-retinol binds with a K(D) = 3.8 nM, and all-trans-retinol (vitamin A) binds with a K(D) = 10.8 nM. This stereospecificity for tRP is to be compared to the binding of retinoids to BSA, where virtually no discrimination is found in the binding of the same retinoids. This work provides further evidence that RPE65 functions by binding to and mobilizing the highly hydrophobic all-trans-retinyl esters, allowing them to enter the visual cycle.     

Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina

11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X [3H]11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark.     

Esterification of retinol in rat liver. Possible participation by cellular retinol-binding protein and cellular retinol-binding protein II

Retinol bound to cellular retinol-binding protein (CRBP) was available for esterification by liver microsomes in the absence of exogenous acyl donors. Moreover, exogenous acyl-CoA gave little or no stimulation of ester production over what was observed with the endogenous acyl donor. In contrast, unbound retinol was esterified in an acyl-CoA-dependent reaction. The presence of two different enzyme activities, acyl-CoA-dependent and -independent, was demonstrated by differential sensitivities to several enzyme inhibitors. The enzyme reaction with retinol-CRBP and endogenous acyl donor produced retinyl esters normally found in vivo in liver. In addition, rates of esterification with this system were sufficient to maintain liver stores. Liver also contains cellular retinol-binding protein, type II (CRBP(II] during the perinatal period. Radioimmunoassay revealed highest levels of CRBP(II) in liver 3-4 days after birth. Examination of retinol esterification by microsomes from the liver of 3-day-old rats revealed a retinyl ester synthase activity with lower Km and higher Vmax than that found in the adult. The activity could use either retinol-CRBP or retinol-CRBP(II) and an endogenous acyl donor. The microsomes from 3-day-old liver had greater esterifying ability than microsomes from adult liver, perhaps due to the presence of two retinyl ester synthase enzymes.     

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.     

Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.     

The complete primary structure of the cellular retinaldehyde-binding protein from bovine retina

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol and 11-cis-retinaldehyde as endogenous ligands and may be a functional component of the visual cycle. The complete amino acid sequence of CRALBP from bovine retina has been determined by direct microanalysis of the protein. Bovine CRALBP contains 316 residues in a single amino-terminal-blocked chain corresponding to a molecular weight of 36,421, inclusive of the blocking group. Overlapping peptides were generated by cleavage of lysyl, arginyl, methionyl, glutamyl, and one tryptophanyl bond and sequenced by gas-phase Edman degradation. Analysis of amino-terminal arginyl and methionyl peptides by fast atom bombardment mass spectrometry identified the N alpha-blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 9 residues. Comparison of CRALBP with other known protein sequences reveals no significant structural relatedness. The present results provide a basis for relating CRALBP domains with physiological function and for the future development of a more detailed three-dimensional model of the interaction of 11-cis-retinaldehyde with protein.