tion in the RPE are 11-c/s retinyl ester hydrolysis, and lecithin: retinol acyltransferase (LRAT) activity. The capacity of LRAT activity to catalyze the formation of ...
Colocalization of 11-cis Retinyl Esters and Retinyl Ester Hydrolase Activity in Retinal Pigment Epithelium Plasma Membrane Nathan L Mata, Elia T. Villazana, and Andrew T. C. Tsin identify the subcellular locale of 11-cis retinyl esters in bovine retinal pigment epithelium (RPE) and to characterize the enzymic mechanism responsible for liberation of 11-cis retinoids in this compartment.
PURPOSE. TO
Endoplasmic reticulum (ER)-enriched and plasma membrane (PM)-enriched protein fractions were prepared from bovine RPE microsomes using sequential discontinuous sucrose and Percoll gradient fractionation. Enzyme markers for ER (such as carboxylesterase), and PM (such as 5'-nucleotidase [5'-ND]; alkaline phosphatase [AP]; and ouabain-sensitive Na+,K+-ATPase [ATPase]) were used to identify the subfractions. Membrane-associated retinoids were quantified by high-performance liquid chromatography (HPLC) and retinyl ester hydrolase (REH) activities were determined by radiometric and chromatographic (HPLC) means. METHODS.
Chromatographic analyses of membrane-associated retinoids showed that 11-cis retinyl esters are localized mainly in PM-enriched fractions, whereas all-trans retinyl esters are associated predominantly with ER-enriched membranes; profiles of the distribution of 11-cis- and all-trans REH activities were consistent with the retinyl ester distribution. Further purification of the crude PM fraction yielded a fraction (P2) that was significantly enriched with 5'-ND (fivefold), ATPase (15-fold), AP (10-fold), and 11-cis retinyl ester hydrolase (11-cis REH; threefold) activities, but was relatively devoid of carboxylesterase and all-trans REH activities. Apparent kinetic constants (^Tmapp and Vmapp) for 11-cis REH activity in P2 were 18 /xM and 1800 picomoles/min per mg, respectively. RESULTS.
CONCLUSIONS. This is the first identification of an 1 1-as-specific REH activity in RPE plasma membrane. Results from these studies demonstrate the capacity of RPE plasma membranes to accommodate and hydrolyze 11-cis retinyl esters. Plasma membrane storage and mobilization of 11-as retinyl esters represents a novel compartmentalization of retinoid metabolism that is distinct from the sites where 11-as retinoids are produced. The implication of these findings for present theories of visual chromophore biosynthesis are discussed. (Invest Ophthalmol Vis Sci. 1998;39: 1312-1319)
B
iosynthesis of 11-as retinoids from dietary vitamin A (all-trans retinol) is a distinctive feature of the visual system, which relies on an isomerohydrolase enzyme in retinal pigment epithelium (RPE) membranes.1'2 The isomerohydrolase enzyme couples the free energy of all-trans retinyl ester hydrolysis to an isomerization reaction to generate 11-as retinol.3 The liberation of 11-c/s retinol represents a branchpoint in the visual cycle in which either 11-as retinal4"6 or 11-a's retinyl esters may be produced.7 It is clear that 11-as retinal biosynthesis in the RPE supports rhodopsin biosynthesis in the retina; however, the role of 11-a's retinyl esters stored in RPE membranes has not been examined.
From the Division of Life Sciences, The University of Texas at San Antonio. Supported by the National Institutes of Health, Bethesda, Maryland (grants EY06438 and GM08194) and the San Antonio Area Foundation. Submitted for publication October 30, 1997; revised February 18, 1998; accepted March 16, 1998. Proprietary interest category: N. Reprint requests: Andrew T. C. Tsin, Division of Life Sciences, The University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, TX 78249-0662.
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There are various lines of evidence that suggest the presence of a regulated mechanism for 11-a's retinyl ester mobilization. For example, newly admitted all-trans retinol is steadily incorporated into the endogenous 11-as retinyl palmitate pool during dark adaptation8; 11-as retinyl ester concentration in the RPE also increases during this period. 910 Moreover, it has been shown that 11-a's retinyl esters are selectively used during light adaptation.10"12 Thus, it is likely that stored 11-a's retinyl esters are used to produce visual chromophore through an accessory metabolic pathway. Two possible mechanisms for 11-a's retinyl ester utilization in the RPE are 11-c/s retinyl ester hydrolysis, and lecithin: retinol acyltransferase (LRAT) activity. The capacity of LRAT activity to catalyze the formation of retinyl esters has been well documented.1314 In addition, LRAT activity also has been shown to catalyze an 11-a's retinyl ester-11-a's retinol exchange reaction in which the palmitate moiety of in situ synthesized 11-a's retinyl palmitate is transferred to 11-a's retinol.15 Thus, LRAT activity seems to have the capacity to synthesize 11-a's retinyl esters using an esterification reaction and also the capacity to liberate 11-a's retinol from the syndiesized 11-a's retinyl ester pool by reversal of the esterification reaction. Alternatively, 11-a's retinol may be liberated using an Investigative Ophthalmology & Visual Science, July 1998, Vol. 39, No. 8 Copyright © Association for Research in Vision and Ophthalmology
IOVS, July 1998, Vol. 39, No. 8
11-cis Retinyl Esters and 11-cis REH Activity in RPE
11-cis retinyl ester hydrolase enzyme. Biochemical properties of 11 -cis retinyl ester hydrolysis have been investigated in human RPE16 and in bovine RPE.17 Studies designed to address the possibility that reversibility of the LRAT reaction (rLRAT) may have been responsible for 11-cis retinol liberation suggested no contribution from rLRAT to the observed 11-cis retinyl ester hydrolase (REH) activity.17 Further investigation of these two mechanisms will be required to reconcile the apparent discrepancies. It is noteworthy that all the key enzymes involved in the visual cycle are intimately associated with membranes. Consequently, purification efforts have been largely unsuccessful, with the exception of a putative 11-cis-specific retinol dehydrogenase.6 This impasse has caused investigators to use microsomal membranes as an enzyme source for biochemical characterizations. In this report, we have addressed the subcellular localization of 11-cis retinyl esters and 11-cis REH activity. Findings from this investigation reveal that 1 l-cis REH activity and 1 l-cis retinyl esters are colocalized in plasma membranes of the RPE. The production of 11-cis retinoids from nil-trans retinol occurs at a site that is spatially separated from the plasma membrane compartment.18 Thus, it is reasonable to suggest that 11-cis retinoids may be mobilized through two distinct subcellular pathways. Although the relationship between these two pathways is not presently clear, it is likely that these processes operate in a complementary fashion to provide visual chromophore for daily photopigment renewal and photopigment regeneration during periods of intense or prolonged light exposure.
1313
Preparation of Subcellular Fractions. AH procedures were performed on ice, or at 4°C. Microsomal protein was prepared from homogenates of fresh RPE according to methods previously described.17 Microsomal proteins were separated immediately on a discontinuous sucrose gradient into plasma membrane (PM)- enriched and endoplasmic reticulum (ER)-enriched fractions following methods described by Touster et al.19 Briefly, microsomal membranes, homogenized in 10 ml Tris-buffered sucrose (57%), were placed at the bottom of a clear polyallomer centrifuge tube followed the addition of 18 ml 34% sucrose and 8 ml 8.5% (0.25 M) sucrose. The sucrose gradient was centrifuged for 22 hours at 75,500g. The resulting gradient fractions (S1-S5, from the top to the bottom of the centrifuge tube) were assayed for PM and ER enzyme markers (i.e., 5'-ND and carboxylesterase, respectively) and for 1 l-cis and all-trans REH activity. Protein fractions were stored at -85°C after dilution to less than 5% sucrose (vol/vol). All protein determinations were made by the dye-binding method using bovine serum albumin as a standard.20
Subfractionation of Plasma Membrane Proteins. Further purification of the PM-enriched fraction generated by discontinuous sucrose density gradient centrifugation (S2) was achieved by using self-forming gradients of Percoll as described by Ottonello and Mariani.21 Optimal resolution of the enzyme markers was achieved when the density of the isoosmotic Percoll-protein solution was 1.045 and the centrifugation was performed at 12,000g for 20 minutes. The recovered fractions (P1-P18) were assayed for protein concentration, 5'-ND, carboxylesterase, ouabain-sensitive Na+,K+-ATPase (ATPase), AP, 1 l-cis, and all-trans REH activities. Enzyme Marker Studies. Carboxylesterase activity was determined according to the methods of Mentlein and HeyMATERIALS AND METHODS mann.22 Briefly, production of onitrophenol was measured spectrophotometrically (420 nm) during the incubation of proMaterials tein samples (10-250 /xg) with o-nitrophenyl acetate (0.18 M All-trans retinyl palmitate, bovine serum albumin, dithiothreistock in ice-cold methanol). Protein and assay buffer (2.7 ml/20 tol, disodium EDTA, all-trans retinol, all-trans retinyl palmitate, mM potassium phosphate, 1 mM EDTA, and 0.1% Triton X-100, 5'-nucleotidase (5'-ND), alkaline phosphatase (AP), and carpH 7.4) were preincubated in a quartz cuvette for 10 minutes boxylesterase assay reagents were purchased from Sigma at 22°C before the addition of o-nitrophenyl acetate (3 mM). Chemical (St. Louis, MO). Percoll was obtained from Pharmacia Product abundance versus time measurements were then Biotech (Uppsala, Sweden). Ouabain was purchased from Retaken every 30 seconds for a period of 10 minutes. Rates were search Biochemicals International (Natick, MA). [9,10corrected for protein concentration and nonenzymatic activity. 3 H] Palmitic acid (specific activity 37 Ci/millimole) was pur5'-ND activity was determined as described in Sigma Procedure chased from DuPont-NEN (Rochester, NY). Purified, unlabeled No. 265-UV23 or Procedure No. 675,24, which describe the 11 -cis retinyl palmitate was a gift from Hoffman-La Roche quantitation of 5'-ND activity through the rate of nicotinamide (Nutley, NJ). All other retinoids were purified by high-perforadenine dinucleotide (NAD) formation and inorganic phosphomance liquid chromatography (HPLC) and quantified by UVrus production, respectively. Spectrophotometric analyses of visible spectrophotometry before use in REH assays. Econo-1 rate versus time (340 nm for NAD production) and singleliquid scintillation cocktail and HPLC grade solvents were obcomponent analysis (660 nm for inorganic phosphorus productained from Fisher Scientific (Houston, TX). Quantitation of tion) were performed. 5'-ND activities were corrected for 3 M [ H] and [ C] was achieved with a liquid scintillation analyzer protein concentration and nonspecific phosphatase activity (model 2200CA; Packard Instrument, Downers Grove, IL). when quantitating inorganic phosphorus. Heat-denatured protein was used to correct for nonenzymatic carboxylesterase Methods and 5'-ND activities. ATPase activity was determined as de25 3 5 Substrate Preparation. [ H] 11-cis and [ H]all-trans reti- scribed by Braunagel et al. 26AP activities were measured using the methods of Bergmeyer. nyl palmitate substrates were prepared by reacting the respective vitamin A alcohols with [9,10-3H]palmitic acid anhydride Quantitation of Retinyl Ester Hydrolase Activity. The as previously described.17 Specific activity of the retinyl ester radiometric REH assay first described by Prystowsky et al.27 was used in the present study. Labeled substrates (i.e., [3H] 11substrates was adjusted to 65,000 to 75,000 dpm/nanomole by the addition of unlabeled 1 l-cis or all-trans retinyl palmitate. cis- or [3H]all-fraras retinyl palmitate) were delivered in a 10-JU,1 volume of ethanol to preincubated reaction mixtures containSubstrate was routinely added to reaction mixtures in 10 /LLI ing 50 mM Tris-acetate, pH 8.0, and protein (final reaction ethanol.
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IOVS, July 1998, Vol. 39, No. 8
Mata et aL
volume = 200 jal). It should be noted that, although the concentration of ethanol in the REH reaction mixtures is relatively high (5%, vol/vol), the effect on the rate of hydrolysis is negligible when compared with REH reaction mixtures in which the ethanol concentration is 0.5% to 1.0%. Thus, the higher ethanol concentration was used because it greatly enhances the solubility of the retinyl ester substrates. After a timed incubation at 37°C, aqueous and lipid phases of the reaction mixtures were partitioned, as described by Belfrage and Vaughan.28 One milliliter of the aqueous ([3H]palmitic acid) phase was removed, was placed in 10 ml Econo-1 scintillation cocktail, and was analyzed for [3H] using a liquid scintillation counter. REH activity is represented as the molar amount of tritiated free fatty acid liberated/minute per milligram of protein. Samples were analyzed in triplicate during each experiment, and nonenzymatic hydrolysis of the substrates was assessed in each analysis using heat-denatured protein. Extraction and High-Performance Liquid Chromatography Analysis of Membrane-Associated Retinyl Esters. Protein samples (5-10 mg protein in a total volume of 1 ml) were mixed with 2 ml absolute ethanol and were left at room temperature for 15 minutes. Retinoids were partitioned into 15 ml of petroleum ether (three 5-ml extractions). The petroleum ether extract was evaporated to dryness under a stream of N2 and then redissolved in 200 to 500 pi 0.2% dioxane/w-hexane. The samples were analyzed isocratically (flow rate = 2 ml/min) by normal-phase HPLC on a silica column (Microsorb; Rainin Instrument, Woburn, MA; 4.6 X 150 mm). Peak absorbance was monitored at 325 nm with a photodiode army detector (model 168; Beckman Instruments, Berkeley, CA); absorption spectra (450-210 nm) for all peaks were obtained simultaneously. Chromatographic data were integrated using PC software (System Gold; Beckman). The analysis of retinyl esters in sucrose gradient fractions was performed similarly after sedimentation of membrane proteins by dilution and ultracentrifugation (150,000g, 60 minutes), or by precipitation with 10% trichloroacetic acid and ultracentrifugation.
RESULTS
Subcellular Locale of 11-cis Retinyl Ester Hydrolase Activity Bovine RPE microsomes were subfractionated on a discontinuous sucrose gradient into five distinct membrane fractions (S1-S5). The membrane fractions were routinely analyzed for esterase (o-nitrophenyl-acetate esterase) and 5'-ND activity to confirm the enzymatic competence of these fractions and to assign a subcellular localization for 11-c/s REH activity. The physical characteristics of the gradient, and the locale of PM and ER enzyme markers, were similar to those reported for subfractionated liver microsomes by Touster et al.19 Thus, the uppermost fraction (SI) contained very little protein and was relatively devoid of all measured enzyme activities (Fig. 1). Fraction S2 appeared as a thick band of white material, which migrated between the 34% (density = 1.15, 5°C) and the 0.25 M sucrose overlayer and contained the largest percentage of 5'-ND activity (48%; Fig. IB). The intermediate fraction (S3) was relatively clear and contained moderate amounts of 5'-ND (20%) and carboxylesterase (30%) activities. The largest per-
Protein
O O
0) i_
0) >
o o 0)
40
All-trans REH
20 S1
til S2
S3
S4
S5
FIGURE 1. Distribution of 11-cis and all-trans retinyl ester hydrolase (REH) activities in sucrose gradient fractions. Freshly prepared bovine retinal pigment epithelium microsomal protein was separated on a discontinuous gradient of sucrose into five discrete protein fractions (S1-S5, from top to bottom of centrifuge tube). Gradient fractions were assayed for protein (A), 5'-nucleotidase (B), carboxylesterase (C), 11-cis REH activity (D), and al\-trans REH activity (E). Conditions for the sucrose-density gradient and for the various enzyme assays are given in the text. In (A), the percentage of protein recovered in each fraction was determined by the amount of protein in that fraction divided by the total amount of protein recovered from all gradient fractions. Similarly, the percentages shown in (B, C, D, E) represent enzyme units in the indicated gradient fractions divided by die total enzyme units recovered in all gradient fractions. In (D) and (E), the total enzyme units recovered from all gradients were higher (up to 40%) than those loaded onto the sucrose gradient, probably as a result of the purification of enzymes from inhibitors. The data shown are mean values of six separate gradient preparations ± SD.
centage of the protein recovered from the gradient was found in fraction S4 (42%; Fig. 1A); the specific activity and total recovery of carboxylesterase activity also was highest in this fraction (44%; Fig. 1C). The lowermost fraction (S5) was similar to SI in that it was devoid of protein and enzyme activities. Analysis of REH activities revealed that 11-cis REH activity was primarily associated with the PM-enriched fraction (49%; Fig. ID), whereas the greatest percentage of all-trans REH activity was recovered in the ER-enriched fraction (36%; Fig. IE). It is noteworthy that the specific activity of 11-cis retinyl
IOVS, July 1998, Vol. 39, No. 8 ester hydrolysis was increased (twofold) in S2 relative to the microsomal value; all-trans REH-specific activity was comparable among fractions S2 to S4. These results, and the finding that approximately 25% of the al\-trans REH activity is present also in fraction S2, prompted our focus on fractions S2 and S4. Subcellular Distribution of 11-cis Retinyl Esters Retinoids were extracted from RPE microsomal protein, fraction S2, and fraction S4 to determine whether a colocalization of REH activity and the respective retinyl ester substrate exists. Data obtained from HPLC analysis of extracts from microsomes, from S2, and from S4 are given in Figure 2 (left side). The identity of the retinyl esters found in these proteins was established by photodiode array absorption spectra (shown on the right) and was confirmed by coelution with authentic retinyl ester standards. The quantitative data are provided in the figure legend. Microsomal protein contained 11-cis- and ail-trans retinyl esters (peak 1 and peak 2, respectively). Peak 2 also contains a retinoid species that demonstrates an absorption spectra that is identical to that of all-trans retinyl palmitate (data not shown). A further investigation of peak 2 using different chromatographic conditions (i.e., 0.1% dioxane/rc-hexane at a 1.5-ml/min flow rate) was effective to further separate the two coeluting retinoid species. The two peaks were collected from the HPLC eluate, mixed with authentic aW-trans retinyl ester standards, and then were resolved on the 0.1% dioxane/n-hexane system. The chromatograms from these analyses indicated that the minor retinoid species was aW-trans retinyl stearate and confirmed that the major component of peak 2 was in fact all-trans retinyl palmitate. The peak that precedes peak 2 in microsomes and S2 was determined to be 9-cis retinyl palmitate. The small peak that precedes peak 1 (retention time = 4.75 minutes) in these fractions was not identified. The localization of 11-cis and all-trans retinyl esters was found to be quite distinct. Using the microsomal 11-cis and all-trans retinyl ester concentrations as a reference (0.70 nanomoles/mg and 1.10 nanomoles/mg, respectively), 67% of the 11-cis retinyl esters were found in S2 and 70% of the all-trans retinyl esters were recovered in S4. Thus, 11-cis retinyl esters seem to be concentrated in a compartment where recovery of 11-cis REH activity is also greatest; a similar trend also was observed for all-trans retinyl esters and all-trans REH activity. The remaining gradient fractions demonstrated relatively insignificant recoveries (\\ iillit-sis
Rliniinnsin Biosynthesis
FIGURE 5. Compartmentalization of vitamin A metabolic pathways in the retinal pigment epithelium (RPE). The diagram shows two metabolic pathways by which visual chromophore may be synthesized. The incorporation of ati-trans retinol bound to serum retinol-binding protein (SRBP) and svibsequent intracellular binding to cellular retinol-binding protein (CRBP) has been well characterized (see Refs. 1 and 7 for review). In the endoplasmic reticulum (ER) pathway, the complete complement of enzymic activities necessary to generate retinyl esters and 1 l-cis retinal are present (i.e., lecithinxetinol acyltransferase [LRAT]; isomerohydrolase [Iso-Hydro]; and 1 l-cis retinol oxidase [1l-cis RO]). It is clear that all-trans retinyl esters serve as a precursor substrate for subsequent synthesis of ll-cis retinal, which presumably is shuttled to apical processes of the RPE conjugated to cellular retinaldehyde-binding protein (CRALBP). ll-c/5 retinyl esters, which are produced from ll-cis retinol using LRAT activity, are localized in the plasma membrane (PM) compartment. Therefore, a presumptive fatty acid-binding protein (FABP, indicated by an asterisk) is thought to facilitate intracellular transport of ll-cis retinyl esters from the site of synthesis (ER) to the PM: where ll-cis REH activity is also localized. Subsequent oxidation using ll-cis RO activity results in the liberation of 1 l-cis retinal directly at the apical RPE, thereby precluding the need for intracellular transport by CRALBP. retinol oxidase activity in RPE PM (unpublished observations). Therefore, it is conceivable that visual chromophore may be generated within the PM compartment. Data from the present report support the hypothesis that ll-cis retinoids may be mobilized from at least two separate subcellular compartments. Observations made by Okajima et al.4'1 during an investigation of the effects of interphotoreceptor retinoid-binding protein on the release of 1 l-cis retinal from amphibian RPE provide direct evidence that is supportive of this hypothesis. Results from the amphibian study clearly demonstrate that a single pool of retinoid precursor could not be responsible for the observed release of ll-cis retinal in the presence of interphotoreceptor retinoid-binding protein. Rather, the existence of discrete pools, or compartments, of nil-trans and 11-c/s retinyl esters, which are differentially mobilized in the presence of interphotoreceptor retinoid-binding protein, would render their findings interpretable. It is conceivable that an acces-
metabolic pathway exists in the RPE to separate the mobilization of the readily available ll-cis retinoid pool (PM pathway) from the mobilization of ll-cis retinal through an all-trans retinyl ester intermediate (ER pathway). A diagram of the metabolic pathways that are theorized to contribute to visual chromophore biosynthesis in the RPE is shown in Figure 5. Proper integration of these pathways (presumably through specific binding proteins) may prove to be vital for photopigment renewal and regeneration. Future studies will be directed toward understanding the relationship of vitamin A metabolism in these subcellular compartments. Acknowledgments The authors thank Jack Saari for comments and suggestions, Clarise Rivera for technical assistance, and Hoffman-La Roche for the contribution of purified 1l-cz's retinyl palmitate.
IOVS, July 1998, Vol. 39, No. 8
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