Rod outer segment retinol dehydrogenase: substrate specificity and role in phototransduction.

The reaction catalyzed by all-trans-retinol dehydrogenase of rod outer segments completes the quenching of photoactivated rhodopsin and initiates the cycle of reactions leading to regeneration of visual pigment. The goal of this study was to determine the kinetic parameters of the dehydrogenase at physiological levels of bleaching, to investigate its specificity, and to determine its possible role in modulating phototransduction. Reduction of all-trans-retinal could be measured after bleaching < 0.15% rhodopsin. Kinetic parameters for the forward reaction determined with endogenous all-trans-retinal were Km = 1.1 microM; Vmax = 7 nmol/min/mg rhodopsin. The low enzymatic activity suggests that at high bleach rates, all-trans-retinal could accumulate, increasing the steady state level of bleaching intermediates or promoting formation of pseudophotoproducts. Active pseudophotoproducts, which stimulate Gt activation and opsin phosphorylation by rhodopsin kinase, are formed with opsin and all-trans-retinal as well as retinal analogues lacking the 13 methyl or the terminal two carbons of the polyene chain. Addition of all-trans-retinol, NADP, and [32P]ATP to rod outer segments increased rhodopsin phosphorylation. Kinetic parameters for the reverse reaction determined with exogenous all-trans-retinol were Km = 10 microM; Vmax = 11 nmol/min/mg rhodopsin. Our results support the hypothesis that all-trans-retinol dehydrogenase could influence the phototransduction cascade, including activities of Gt, rhodopsin kinase, and binding of arrestin, by impeding the recycling of rhodopsin at high bleach levels.

Control of substrate flow at a branch in the visual cycle.

Photoisomerization of rhodopsin's chromophore, 11-cis-retinaldehyde, and subsequent regeneration of the 11-cis configuration are accomplished in vertebrates by a series of reactions known as the visual cycle. At one point in the cycle, 11-cis-retinol can either be enzymatically oxidized to 11-cis-retinaldehyde and exported for visual pigment regeneration or be enzymatically esterified and stored. Partition of substrate at this branch was examined in this study and found to be influenced by cellular retinaldehyde-binding protein (CRALBP), a retinoid-binding protein found in retina. Esterification was reduced to about 10% and oxidation stimulated 2-3-fold in the presence of this protein. Other experiments confirmed that "free" 11-cis-retinol was esterified more rapidly than 11-cis-retinol complexed with CRALBP and that CRALBP.11-cis-retinol was not an inhibitor of the esterification. Following oxidation of CRALBP.11-cis-retinol, the reaction product, 11-cis-retinaldehyde, was found associated with the binding protein. 11-cis-Retinaldehyde is not available for reaction with carbonyl reagents when the retinoid is bound to CRALBP. However, enzymatic oxidation of CRALBP.11-cis-retinol in the presence of O-ethylhydroxylamine produced ca. 30% retinaldehyde O-ethyloxime and 70% free 11-cis-retinaldehyde, suggesting that about one-third of the retinol oxidized had dissociated from the binding protein. Neither oxidation nor esterification of CRALBP.11-cis-retinol was inhibited by including CRALBP.11-cis-retinaldehyde in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Assays of retinoid dehydrogenases by phase partition.

Two modifications of an extraction assay for retinoid dehydrogenases are described. The first method involves the transfer of tritium from carbon-15 of [15-3H]-retinol or [15-3H]retinaldehyde to NAD, whereas in the second method, tritium from [3H]NADPH is transferred to all-trans-retinaldehyde. Since both versions of the assay involve the interconversion of water-soluble and -insoluble tritium-labeled compounds, a simple phase partition is sufficient to separate labeled products from labeled reactants. The assays are shown to provide reliable estimations of the reaction progress for three retinoid dehydrogenases of the visual system when compared to HPLC analysis of retinoid products or gel filtration analysis of pyridine nucleotide. The assays will be useful in studying retinoid dehydrogenases from other tissues and in principle can be modified for other dehydrogenase reactions with water-insoluble substrates.

Retinol esterification in bovine retinal pigment epithelium: reversibility of lecithin:retinol acyltransferase.

Esterification of all-trans-retinol is a key reaction of the vertebrate visual cycle, since it produces an insoluble, relatively non-toxic, form of the vitamin for storage and supplies substrate for the isomerization reaction. CoA-dependent and -independent pathways have been described for retinol esterification in retinal pigment epithelium (RPE). The CoA-independent reaction, catalysed by lecithin:retinol acyltransferase (LRAT) was examined in more detail in this study. Addition of retinol to RPE microsomes results in a burst of retinyl ester synthesis, followed by a rapid apparent cessation of the reaction. However, [3H]retinol, added when retinyl ester synthesis has apparently ceased, is rapidly incorporated into retinyl ester without a net increase in the amount of ester. The specific radioactivities of [3H]retinol and [3H]retinyl ester reach the same value. [14C]Palmitate from palmitoyl-CoA is incorporated into preexisting retinyl ester in the absence of net ester synthesis, too. These exchange reactions suggest that the reaction has reached equilibrium at the plateau of the progress curve and that only the accumulation of retinyl ester, and not its synthesis, has stopped during this phase of the reaction. Studies with geometrical isomers of retinol revealed that the rate of exchange of all-trans-retinol with all-trans-retinyl esters was about 6 times more rapid than exchange of 11-cis-retinol with 11-cis-retinyl ester. This is the first demonstration of the reversibility of LRAT and the first example of stereospecificity of retinyl ester synthesis in the visual system. Reversal of the LRAT reaction could contribute to the mobilization of 11-cis-retinol from 11-cis-retinyl ester pools.

Lecithin:retinol acyltransferase in retinal pigment epithelial microsomes.

Microsomal preparations from retinal pigment epithelium carry out phosphatidylcholine synthesis upon incubation with 1-palmitoyllysophosphatidylcholine and fatty acyl-CoA. Phosphatidylcholine synthesized in situ in this manner is an acyl donor for retinyl ester synthesis, demonstrating the existence of lecithin:retinol acyltransferase. Although acyl transfer to retinol is from the 1-position of phosphatidylcholine, the fatty acid in the 2-position is important in substrate recognition. The finding of this novel enzyme activity in retinal pigment epithelial microsomes suggests that phosphatidylcholine is the endogenous acyl donor in CoA-independent retinol esterification observed in these preparations.

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.

Purification of cellular retinaldehyde-binding protein from bovine retina and retinal pigment epithelium.

Cellular retinaldehyde-binding protein (CRALP) has been purified from extracts of bovine retina or retinal pigment epithelium by a procedure employing an initial, high-capacity anion exchange chromatographic step and anion exchange HPLC for removal of a persistent contaminant. The procedure also yields fractions containing three other retinoid-binding proteins present in retina (cellular retinol-, cellular retinoic acid- and interphotoreceptor retinol-binding proteins; CRBP, CRABP and IRBP, respectively). Procedures are described for labeling CRALBP with 9-cis-retinaldehyde, 11-cis-retinaldehyde, or 11-cis-retinol. There are approx. 3 nmol of CRALBP per adult bovine eye and the binding protein is ca. 0.5% of the soluble protein of a retinal supernatant.

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.

Characterization of the interstitial space: immunocytochemical and biochemical studies.

The interstitial space has been characterized using immunocytochemical and biochemical techniques. The pore size of the external limiting membrane that forms the vitread margin of this extracellular space has been probed using a series of biotinylated proteins of known Stokes' radius, followed by avidin-horseradish peroxidase histochemistry. The pore size of the zonulae adherentes that collectively comprise the external limiting membrane has been found to lie between 30 and 36A. This is sufficiently small to block passage of interstitial retinol-binding protein (IRBP, Stokes' radius = 55A) from the interstitial space vitread into the retina. IRBP has been localized to the interstitial space of bovine retina by use of indirect immunogold electron microscopy. IRBP has also been identified in cytoplasmic vesicles of rod and cone inner segments. The function of these vesicles may be for secretion and/or uptake of IRBP from the interstitial space. A postulated function of IRBP is transfer of vitamin A between the retinal pigment epithelium and photoreceptors during the visual cycle. Biochemical experiments have corroborated previous studies that IRBP becomes loaded with all-trans-retinol following bleaching illumination, while the amount of endogenous 11-cis-retinol remains unchanged. These data are consistent with the emerging concept that IRBP plays a role in transport of vitamin A among the cell types that border the interstitial space.

Properties and immunocytochemical localization of three retinoid-binding proteins from bovine retina.

Cellular retinal-binding protein (CRALBP) complexed with 11-cis-retinal has several properties characteristic of a visual pigment. Interaction of the protein and retinoid results in a bathochromic shift in the absorption spectrum of the chromophore from 380 to 425 nm, accompanied by a decrease in the extinction coefficient (25,000-15,000 M-1 cm-1). Illumination of the complex results in the progressive loss of absorbance at 425 nm and an increase at 375 nm, consistent with the production of a geometrical isomer of retinal that lacks affinity for the binding protein. Analysis by HPLC of the retinoids after illumination reveals that the basis of the spectral transition is a photoisomerization of 11-cis-retinal to all-trans-retinal. Only small amounts (less than 10%) of 13-cis-retinal are produced during the photoisomerization, showing the stereospecificity of the process. Although CRALBP has the spectral characteristics of a blue-sensitive visual pigment, there is no evidence that this is related to its function. This protein may serve as a model for the interactions of 11-cis-retinal and protein. Eleven-cis-retinol bound to CRALBP is a better substrate for esterification by microsomes from retinal pigment epithelium (RPE) than all-trans-retinol bound to cellular retinol-binding protein (CRBP). The product of the reaction, retinyl ester, does not remain bound to either binding protein but becomes associated with the microsomal fraction. Esterification is the first described process, occurring in the dark, by which retinoids can be removed from CRBP and CRALBP. Antibodies to bovine CRBP have been produced in rabbits following injection of the performic acid-oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)