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• W2138860259 abstract "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 isomerizationin 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. 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 isomerizationin 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. Photoisomerization of 11-cis-retinal to all-trans-retinal is the key reaction that initiates vision (1Wald G. J. Gen. Physiol. 1935; 19: 351-371Crossref PubMed Scopus (137) Google Scholar). 11-cis-retinal is coupled via a Schiff base to a Lys residue located in the transmembrane portion of the rod and cone photoreceptor opsins. Photoisomerization triggers conformational changes in these receptors that lead to activation of G-proteins and subsequent initiation of the signaling cascade of reactions, which comprise phototransduction (2Polans A. Baehr W. Palczewski K. Trends Neurosci. 1996; 19: 547-554Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). How all-trans-retinal is isomerized back to 11-cis-retinal is one of the fundamental questions in vision. A pulse of intense light, which occasionally bleaches nearly 100% of our visual pigment, quickly generates 3 mmall-trans-retinal in the photoreceptor cell outer segment. Yet in a matter of minutes (the time constant in humans is 400 s for rhodopsin; Ref. 3Alpern M. Maaseidvaag F. Ohba N. Vision Res. 1971; 11: 539-549Crossref PubMed Scopus (54) Google Scholar), the entire cycle of isomerization and pigment regeneration occurs. No free retinals accumulate; all-trans-retinal is either complexed with opsin or reduced and esterified with fatty acids, whereas 11-cis-retinal combines with opsins. Analysis of the visual cycle in mice showed that the concentrations of free retinols and retinals are low compared with the ester pool and with all-trans-retinal and 11-cis-retinal bound to opsins (4Saari J.C. Garwin G.G. Van Hooser J.P. Palczewski K. Vision Res. 1998; 38: 1325Crossref PubMed Scopus (115) Google Scholar). Mutations in any of the genes involved in retinoid metabolism could result in retinal dystrophies and degeneration of photoreceptors. Thus, it is important to understand this metabolic pathway at the molecular level. Understanding of the visual cycle presents several intellectual problems. How does the stereospecific, energy-requiring isomerization occur in a two-cell system, with a substrate that is virtually insoluble in the aqueous phase? By what mechanism does the isomerization product, 11-cis-retinal, leave the retinal pigment epithelial (RPE) 1The abbreviations used are:RPE, retinal pigment epithelial; LRAT, lecithin:retinol acyltransferase; apo-rCRALBP, apo-recombinant cellular retinaldehyde-binding protein; MOPS, 3-N-morpholinopropanesulfonic acid; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin; BTP, 1,3-bis[tris(hydroxymethyl)-methylamino]propane.cells and go to the photoreceptor cells where regeneration of visual pigments occurs? What methods are available to characterize the low abundance, membrane-bound enzymes responsible for retinoid regeneration? It is well established that all-trans-retinal, the product of photoisomerization, is reduced to all-trans-retinol in photoreceptor cells before it diffuses to RPE cells (5Dowling J.E. Nature. 1960; 188: 114-118Crossref PubMed Scopus (323) Google Scholar, 6Zimmerman W.F. Vision Res. 1974; 14: 795-802Crossref PubMed Scopus (100) Google Scholar). It is possible that esterification of all-trans-retinol with fatty acids, catalyzed by lecithin:retinol acyltransferase (LRAT; Fig. 1, reaction 2) in RPE cells, drives this transcellular diffusion by mass action. Rando and colleagues (7Deigner P.S. Law W.C. Canada F.J. Rando R.R. Science. 1989; 244: 968-971Crossref PubMed Scopus (155) Google Scholar) proposed that all-trans-retinyl carboxylic esters are substrates for a membrane-bound isomerohydrolase that catalyzes the reaction in which all-trans- to 11-cis-isomerization is coupled to ester hydrolysis (Fig. 1,reaction 5). 11-cis-retinol formed by this reaction is oxidized to 11-cis-retinal by an NAD/NADP-dependent 11-cis-retinol dehydrogenase(s) present in RPE cells (8Zimmerman W.F. Lion F. Deamen F.J.M. Bonting S.L. Exp. Eye Res. 1975; 21: 325-332Crossref PubMed Scopus (50) Google Scholar) (Fig. 1, reaction 9 and reverse reaction 10) and transported back to the photoreceptor cells where it associates with opsins to form rhodopsins (5Dowling J.E. Nature. 1960; 188: 114-118Crossref PubMed Scopus (323) Google Scholar, 9Brown P.K. Wald G. J. Biol. Chem. 1956; 222: 865-877Abstract Full Text PDF PubMed Google Scholar). 11-cis-retinol can also be esterified by LRAT to 11-cis-retinyl esters (Fig. 1,reaction 8). In addition to this pathway, cones may regenerate their pigments via enzymatic processes that also involve the neural retina (10Goldstein E.B. Vision Res. 1967; 7: 837-845Crossref PubMed Scopus (62) Google Scholar, 11Goldstein E.B. Vision Res. 1970; 10: 1065-1068Crossref PubMed Scopus (52) Google Scholar, 12Saari J.C. Sporn M.B. Roberts A.B. Goodmann D.S. The Retinoids: Biology, Chemistry, and Medicine. 2nd Ed. Raven Press, New York1994: 351-385Google Scholar). What evidence supports the proposed isomerohydrolase reaction in RPE microsomes? 1) Isomerization occurs at the alcohol oxidation state (13Bernstein P.S. Rando R.R. Biochemistry. 1986; 25: 6473-6478Crossref PubMed Scopus (54) Google Scholar,14Bernstein P.S. Law W.C. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1849-1853Crossref PubMed Scopus (161) Google Scholar). 2) Isomerization occurs enzymatically without an exogenous source of energy (14Bernstein P.S. Law W.C. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1849-1853Crossref PubMed Scopus (161) Google Scholar, 15Fulton B.S. Rando R.R. Biochemistry. 1987; 26: 7938-7945Crossref PubMed Scopus (49) Google Scholar). Hydrolysis of all-trans-retinyl carboxylic ester was proposed (7Deigner P.S. Law W.C. Canada F.J. Rando R.R. Science. 1989; 244: 968-971Crossref PubMed Scopus (155) Google Scholar) to provide the energy needed for isomerization (4 kcal/mol; Ref. 16Rando R.R. Chang A. J. Am. Chem. Soc. 1983; 105: 2879-2882Crossref Scopus (74) Google Scholar). 3) The inhibition of all-trans-retinol esterification by an LRAT inhibitor prevents biosynthesis of 11-cis-retinol, which led to the conclusion that retinyl esters are essential intermediates in 11-cis-retinol formation (17Trehan A. Canada F.J. Rando R.R. Biochemistry. 1990; 29: 309-312Crossref PubMed Scopus (65) Google Scholar). 4) During isomerization,18O-labeled all-trans-retinol loses its oxygen. In order for this to happen, the bond between C15 and oxygen must be cleaved. The proposed mechanism of isomerization resembles a base catalyzed SN1 alkyl cleavage. In this mechanism, double bond migration and expulsion of the carboxylate occur during addition of a base to C11 of the retinyl ester. Rotation about the C11-C12 single bond is followed by attack of water at C15 and concomitant reshuffling of the double bonds, which locks the retinol in the 11-cis configuration (7Deigner P.S. Law W.C. Canada F.J. Rando R.R. Science. 1989; 244: 968-971Crossref PubMed Scopus (155) Google Scholar). Despite these studies supporting the isomerohydrolase reaction, there are several puzzling features. 1) Even under the best conditions, isomerization activity is too slow (1.3 pmol/min/mg protein; Ref. 15Fulton B.S. Rando R.R. Biochemistry. 1987; 26: 7938-7945Crossref PubMed Scopus (49) Google Scholar) by a factor >100 to account for the amount of 11-cis-retinol produced by this tissue. 2) Enzymatic production of 11-cis-retinol from all-trans-retinyl ester has not been demonstrated in a chemically defined system (13Bernstein P.S. Rando R.R. Biochemistry. 1986; 25: 6473-6478Crossref PubMed Scopus (54) Google Scholar, 14Bernstein P.S. Law W.C. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1849-1853Crossref PubMed Scopus (161) Google Scholar). 3) Enzymatic endothermic isomerization could be driven by mass action if the 11-cis-retinol product is selectively removed from RPE cells. 4) RPE microsomes contain a high concentration of all-trans-retinyl esters (>50%), which are not converted to 11-cis-retinol despite a favorable ΔG. 5) Evidence for participation of retinyl esters in isomerization is indirect. The LRAT inhibitor employed to block retinyl ester formation (17Trehan A. Canada F.J. Rando R.R. Biochemistry. 1990; 29: 309-312Crossref PubMed Scopus (65) Google Scholar) could also block formation of an unidentified intermediate different from all-trans-retinyl ester. 6) The stoichiometry of the isomerohydrolase reaction has not been established. 7) A putative isomerohydrolase has defied purification and molecular characterization. It was recently shown that apo-recombinant cellular retinaldehyde-binding protein (apo-rCRALBP), which binds 11-cis-retinol and 11-cis-retinal, but not all-trans-retinol and all-trans-retinal (12Saari J.C. Sporn M.B. Roberts A.B. Goodmann D.S. The Retinoids: Biology, Chemistry, and Medicine. 2nd Ed. Raven Press, New York1994: 351-385Google Scholar) or albumin, enhances production of 11-cis-retinol in RPE microsomes (18Winston A. Rando R.R. Biochemistry. 1998; 37: 2044-2050Crossref PubMed Scopus (80) Google Scholar). This suggests that 11-cis-retinol may act as a potent inhibitor of the isomerase. As shown in the current study, we have also found that apo-rCRALBP greatly promotes retinoid isomerization, which allowed us to carefully study isomerization of all-trans-retinol in RPE microsomes. Our data reveal the complexity of the enzymatic activities in RPE microsomes that utilize retinoids. They also reveal several inconsistencies or oversimplifications in the model in which all-trans-retinyl ester is a substrate for a putative isomerohydrolase. Fresh bovine eyes were obtained from a local slaughterhouse (Schenk Packing Co., Inc., Stanwood, WA). Liver microsomes were isolated from fresh bovine liver employing sucrose density gradients as described for isolation of bovine rod outer segments (19Papermaster D. Methods Enzymol. 1982; 81: 48-52Crossref PubMed Scopus (260) Google Scholar). The coding sequence for 11-cis-retinol dehydrogenase (20Simon A. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1995; 270: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) was amplified from bovine retina cDNA by polymerase chain reaction with primer FH51 (5′-CATATGTGGCTGCCTCTGCTGCTG-3′), which placed a NdeI restriction site at the ATG initiation site, and primer FH52 (5′-TTAGTAGACTGTCTGGGCAGG-3′) by 32 cycles at 94 °C for 30 s and 68 °C for 2.5 min. The polymerase chain reaction fragment was cloned into the pCRTM 2.1 vector (TA cloning kit, Invitrogen) (designated pFR425) and sequenced by dyedeoxy terminator sequencing (ABI-Prism, Perkin-Elmer). The expression cassette was then transferred into the baculovirus shuttle vector (bacmid) by transposition. Sf9 insect cells were transfected with the recombinant bacmid using cationic liposome-mediated transfection (CellFECTIN reagent, Life Technologies Inc.) according to the manufacturer's protocol. For the expression of recombinant proteins, cells cultured in Sf-900 II SFM medium (Life Technologies, Inc.) at 27 °C were harvested by centrifugation at 1200 × g, 72–96 h after infection (21Haeseleer F. Huang J. Lebioda L. Saari J.C. Palczewski K. J. Biol. Chem. 1998; 273: 21790-21799Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). A microsomal membrane fraction was obtained from fresh bovine RPE cells as described previously (22Saari J.C. Bredberg D.L. Methods Enzymol. 1990; 190: 156-163Crossref PubMed Scopus (26) Google Scholar). The microsomal fraction was resuspended in 10 mm MOPS, pH 7.0, containing 1 μm leupeptin, and 1 mmdithiothreitol to a total protein concentration of 3.2 mg/ml determined according to the Bradford method (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (219357) Google Scholar), and stored in small aliquots at −80 °C. SDS-polyacrylamide gel electrophoresis analysis showed a typical composition of proteins as observed by others (20Simon A. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1995; 270: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 24Barry R.J. Canada F.J. Rando R.R. J. Biol. Chem. 1989; 264: 9231-9238Abstract Full Text PDF PubMed Google Scholar). The protein pattern did not vary significantly between preparations. Significant variations in enzymatic activities of RPE microsome preparations were observed as a function of length of storage at −80 °C. For example, ester hydrolase and isomerase activities declined by ∼50% during 3 months. Thus, <1-month-old preparations were used for all studies. To destroy endogenous retinoids, RPE microsomes (200-μl aliquots) were irradiated in a quartz cuvette for 5 min at 0 °C using a ChromatoUVE-transilluminator (model TM-15 from UVP Inc.). UV treatment produced RPE microsomes without detectable amounts of all retinoids. Interphotoreceptor retinoid-binding protein was prepared from bovine retinas as described previously (25Saari J.C. Bredberg D.L. Exp. Eye Res. 1988; 46: 569-578Crossref PubMed Scopus (32) Google Scholar). Apo-rCRALBP was expressed in Escherichia coli and purified to apparent homogeneity by Ni2+affinity chromatography as described by Crabb et al. (26Crabb J.W. Chen Y. Goldflam S. West K. Kapron J. Redfern C.P.F. Methods in Molecular Biology : Retinoid Protocols. 89. Humana Press, Totowa, NJ1998: 91-104Google Scholar). Apo-recombinant cellular retinol-binding protein type I was a generous gift from Dr. David Ong (Vanderbilt University, Nashville, TN). To prevent isomerization and oxidation, all procedures involving retinoids were performed under dim red illumination, and the retinoids were stored under argon at −80 °C. [11,12-3H(N)]All-trans-retinol (NEN Life Science Products) was diluted with all-trans-retinol to give the desired specific radioactivity (550,000 dpm/nmol), and purified on a normal phase HPLC column (Altex, Ultrasphere-Si 5u; 4.6 × 250 mm; flow rate, 1.4 ml/min; 10% ethyl acetate in hexane; Ref. 27Landers G.M. Olson J.A. J. Chromatogr. 1988; 438: 383-392Crossref PubMed Scopus (49) Google Scholar). Purified material was dried under argon and stored in vials (0.5–10-nmol aliquots at −80 °C) for up to 3 months. [15-3H(N)]11-cis-retinol (260,000 dpm/nmol) was prepared by reduction of 11-cis-retinal with [3H]NaBH4 and purified by HPLC (28Bridges C.D.B. Fong S.-L. Alvarez R.A. Vision Res. 1980; 20: 355-360Crossref PubMed Scopus (30) Google Scholar). Retinoid concentrations in ethanol were determined spectrophotometrically: 11-cis-retinal, 380 nm, ε = 24,935m−1 cm−1; all-trans-retinal, 383 nm, ε = 42,880m−1 cm−1; 11-cis-retinol, 319 nm, ε = 34,890m−1 cm−1; all-trans-retinol, 325 nm, ε = 52,770m−1 cm−1. Rhodopsin concentration was measured as described by McDowell (29McDowell J.H. Methods Neurosci. 1993; 15: 123-130Crossref Scopus (57) Google Scholar). Substrate, [3H]all-trans-retinol, or [3H]11-cis-retinol (0.5–1 nmol, 260,000–550,000 dpm/nmol), was dried from hexane/ethyl acetate under argon in a 1.5-ml polypropylene tube. 20 μl of BSA (5% solution in 10 mm BTP, pH 7.0) was added followed by 20–30 μl of apo-rCRALBP in 10 mm BTP, pH 8.0, containing 250 mm NaCl to give a final concentration of 25 μm. Next, 10 mm BTP, pH 8.0, containing 100 mm NaCl, 2 mm MgCl2, 2 mm CaCl2 was added to bring the total volume to 175 μl. For some experiments, additional compounds in the same buffer were added. Finally, 25 μl of RPE microsomes (80 μg of protein) were added to this mixture, and the reactions were incubated at 37 °C for the indicated times. The reaction mixture (180 μl of 200 μl) was transferred to a new vial containing 300 μl of ice-cold methanol, and 300 μl of hexane was added. The sample was vortexed for 2 min and centrifuged for 4 min at 14,000 ×g to separate organic and aqueous phases. 10 μl of hexane extract was injected into the HPLC column. Retinoids were separated using an HP1050 HPLC (with a single wavelength detector at 325 nm) or an HP1100 HPLC (with a diode array detector 280–400 nm) and a normal phase, narrow bore column (Alltech, Silica 5μ Solvent Miser, 2.1 × 250 mm). An isocratic solvent composed of 10% ethyl acetate in hexane at a flow rate of 0.3 ml/min was used. All-trans-retinol and 11-cis-retinol were extracted with hexane in 75–95% yield as determined using [3H]retinol tracers. To estimate the yield of retinyl ester extraction, [3H]all-trans-retinol was incubated for 1 h with RPE microsomes as a source of LRAT. Most of the [3H]all-trans-retinol was converted to hydrophobic [3H]all-trans-retinyl esters, which were extracted in ∼60% yield. All-trans-retinyl esters and 11-cis-retinyl esters were eluted 0.5 min after the solvent front (Fig. 2 A, peak 1), followed by 11-cis-retinol (peak 3) and all-trans-retinol (peak 4), all with a chromatographic yield of >95%. 9-cis-retinol eluted ∼1 min earlier than all-trans-retinol, while 13-cis-retinol eluted on the descending side of the 11-cis-retinol peak (data not shown). Without preincubation, native RPE microsomes contained 0–0.9 nmol of all-trans-retinol/mg of protein, 0–0.3 nmol 11-cis-retinol, 8.6 ± 0.9 nmol all-trans-retinyl esters, and 9.6 ± 1.4 nmol 11-cis-retinyl esters. The amounts of all-trans-retinyl esters and 11-cis-retinyl esters were measured after separation of a mixture of esters from polar retinoids, saponification, and another round of HPLC separation to quantify all-trans-retinol and 11-cis-retinol. The different preparations of RPE microsomes had similar amounts of retinols and retinals; however, they differed in amounts of endogenous esters (with similar ratio between 11-cis-retinyl and all-trans-retinyl esters). In some cases, the ester pool was as high as ∼60 nmol/mg of protein. Isomers of retinal were converted to more polar and less chemically reactive oximes before analysis (32Bridges C.D.B. Alvarez R.A. Methods Enzymol. 1982; 81: 463-485Crossref PubMed Scopus (96) Google Scholar). Reaction mixtures were stopped with the addition of an equal volume of methanol containing sufficient NH2OH to give a final concentration of 10 mm. After 30 min at room temperature, 0.3 ml of hexane was added, and retinoids were extracted as described before. Retinals in RPE microsomes were converted to oximes with >85% yield. The syn-conformer of retinal oximes eluted between the retinyl ester and 11-cis-retinol peaks (Fig. 2 A, peak 2), whereas smaller peaks of the anti-conformer eluted on the descending side of the all-trans-retinol peak (descending site of peak 4in Fig. 2 A). Retinals were present in RPE microsomes at a low level (<0.1 nmol/mg of RPE protein) and were not generated in most of our experimental conditions. Hexane from the extracts containing retinyl esters or from the HPLC purified retinyl ester fractions (typically 200 μl) were evaporated under argon. The residual esters were dissolved in 230 μl of absolute ethanol, and 20 μl of 6 m KOH was added. The sample was incubated for 30 min at 55 °C, diluted with 100 μl of water, chilled on ice for 2 min, and extracted with 300 μl of hexane. The retinoids in hexane were analyzed directly by HPLC. SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (208094) Google Scholar) using 12% SDS acrylamide gels in a Hoefer minigel apparatus and low molecular weight markers from Amersham Pharmacia Biotech. The gels were stained with Coomassie Brilliant Blue R-250 and destained with 50% methanol and 7% acetic acid. To test the influence of apo-rCRALBP on formation of 11-cis-retinol from endogenous retinoids, the binding protein was added to a suspension of bovine RPE microsomes and incubated at 37 °C. Retinoids were extracted with hexane and separated on a normal phase silica column under isocratic conditions (Fig. 2 A). Addition of apo-rCRALBP to bovine RPE microsomes resulted in formation of 11-cis-retinol (Fig.3 A, peak 3). To prove that the fraction, which eluted at ∼10 min, contained 11-cis-retinol and not 13-cis-retinol, which co-elutes, 1) UV spectra were recorded continuously during the chromatography (data not shown) and after the fraction was collected. The spectrum showed the smooth ascending and descending limbs (Fig.2 B) characteristic of 11-cis-retinol (31Knowles A. Dartnall H.J.A. Davson H. The Eye. 2B. Academic Press, New York1977: 118Google Scholar) and 2) material in the 10-min fraction was oxidized to the aldehyde by recombinant 11-cis-retinol dehydrogenase and incubated with opsin, giving the characteristic spectrum of rhodopsin (498 nm absorption maximum) with a yield of 85% (Fig. 2 C). Note that 13-cis-retinal does not regenerate opsin. We conclude that the 10-min fraction contained >85% 11-cis-retinol. The retinol was bound to soluble apo-rCRALBP as determined after pelleting the RPE microsomes and analyzing the retinol content of the membrane and supernatant phases. When apo-rCRALBP was boiled or omitted, only 11-cis-retinol endogenous to RPE microsomes was found (Fig. 3, B and C). Boiling the RPE microsomes led to even smaller amounts of 11-cis-retinol and 11-cis-retinyl esters (Fig. 3 D) than in unboiled samples without apo-rCRALBP, presumably due to partial destruction of 11-cis-retinoids. Ester analysis showed an apparent ∼20% increase in the amount of 11-cis-retinyl ester in the sample with apo-rCRALBP as compared with the sample that lacked this retinoid-binding protein. This was attributed to higher extraction yields of polar retinoids, as compared with the more hydrophobic esters. As will be described below, most of the 11-cis-retinol formed in the presence of apo-rCRALBP is due to hydrolysis of 11-cis-retinyl esters. To follow the fate of exogenous all-trans-retinol, RPE microsomes were incubated with 2.5 μm[3H]all-trans-retinol. In the first 5 min of the reaction, more than 90% of [3H]all-trans-retinol was converted to [3H]all-trans-retinyl esters by endogenous LRAT (Fig. 4 A). The amount of [3H]all-trans-retinyl esters decreased during reaction as more [3H]11-cis-retinol was formed in the samples containing apo-rCRALBP (Fig. 4 A). During the course of the reaction, the amount of free [3H]all-trans-retinol decreased rapidly to ∼0.12 μm (or ∼0.3 nmol/mg of protein). The decrease in the ester pool coincides with an increase in 11-cis-retinol, suggesting a precursor-product relationship (13Bernstein P.S. Rando R.R. Biochemistry. 1986; 25: 6473-6478Crossref PubMed Scopus (54) Google Scholar, 14Bernstein P.S. Law W.C. Rando R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1849-1853Crossref PubMed Scopus (161) Google Scholar). However, evidence to be presented under “UV-treated RPE Microsomes” indicates that only a small fraction of the ester pool, if any, participates in the isomerization reaction. Thus, it is more likely that the decrease in the ester pool reflects conversion to all-trans-retinol, subsequent isomerization to 11-cis-retinol, and binding to apo-rCRALBP. Based on hydrolysis of the ester pool and rechromatography of retinols, no detectable amounts of [3H]11-cis-retinyl esters were formed during the reaction (data not shown). Isomerization was measured as the formation of [3H]11-cis-retinol. Based on the initial rate, the isomerization proceeded with formation of 0.09 nmol of 11-cis-retinol/min/mg of protein (Fig.4 A). 2Because RPE microsomes contain endogenous retinoids, the specific radioactivity of the labeled pool is lower than that of the [3H]all-trans-retinol added. The mol amounts given for each retinoid species are calculated from the cpm data using the specific radioactivity of the added [3H]all-trans-retinol and are lower limit values. The amount of [3H]all-trans-retinol converted to [3H]13-cis-retinol was estimated as ∼5 pmol/mg of protein by quantifying the amount of [3H]13-cis-retinol formed at time 0 in samples with boiled apo-rCRALBP. 13-cis-retinol was identified by its specific retention time (0.2 min after 11-cis-retinol) and characteristic UV spectrum (data not shown) (31Knowles A. Dartnall H.J.A. Davson H. The Eye. 2B. Academic Press, New York1977: 118Google Scholar). No additional [3H]13-cis-retinol was formed during incubation with RPE microsomes (data not shown). Thus, 13-cis-retinol was only a small fraction (∼1/200) of 11-cis-retinol and did not significantly affect interpretation of our data. In addition to isomerization, 11-cis-retinol was also produced as a result of hydrolysis of endogenous 11-cis-retinyl esters (described below in Fig. 5). The isomerization plus hydrolysis reactions proceeded with an initial rate of 1.4 nmol/min/mg of protein, which is faster than the rate of isomerization (0.09 nmol of 11-cis-retinol/min/mg of protein), and reached a plateau in ∼60 min to form ∼16 nmol of 11-cis-retinol/mg of RPE protein (Fig. 4 A). As shown in Fig. 4 B, the concentration of apo-rCRALBP used in the above experiments (25 μm) was sufficient to give maximal effects on 11-cis-retinol and [3H]11-cis-retinol formation. Note that the concentration of CRALBP used in this study is within the in vivo concentration predicted to be ∼25–50 μm(12Saari J.C. Sporn M.B. Roberts A.B. Goodmann D.S. The Retinoids: Biology, Chemistry, and Medicine. 2nd Ed. Raven Press, New York1994: 351-385Google Scholar). The initial formation of [3H]all-trans-retinyl esters was unaffected by the presence of active apo-rCRALBP or boiled apo-rCRALBP. To measure retinyl ester hydrolase activity directly without isomerization, exogenously added [3H]11-cis-retinol was converted to 11-cis-retinyl esters, by LRAT present in RPE microsomes, before apo-rCRALBP was added. Most of the exogenously added 11-cis-retinol (∼90%) was converted to the esters. Addition of apo-rCRALBP led to the release of 11-cis-retinol and [3H]11-cis-retinol (Fig.5, inset A) with an initial rate of 0.7 nmol/min/mg of protein. If isomerization would contribute significantly to the total amounts of 11-cis-retinol formed, it is expected that with time, more 11-cis-retinol would be converted, diluting [3H]11-cis-retinol formed from the [3H]11-cis-retinyl ester pool. The ratio of [3H]11-cis-retinol to total 11-cis-retinol, however, remained constant during the reaction (Fig. 5, inset B), suggesting only a small contribution of the isomerization of endogenous all-trans-retinol to 11-cis-retinol formation. No radioactivity was detected in the all-trans-retinol peak in the presence or absence of apo-rCRALBP. All together, the results show that isomerization of all-trans-retinol to 11-cis-retinol is essentially an irreversible reaction in RPE microsomes under the conditions studied. In control experiments, liver microsomes were used to assay isomerase activity in the presence of apo-rCRALBP and exogenously added [3H]" @default.
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• W2138860259 date "1999-03-01" @default.
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• W2138860259 title "Preferential Release of 11-cis-retinol from Retinal Pigment Epithelial Cells in the Presence of Cellular Retinaldehyde-binding Protein" @default.
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