Immunochemical Distribution of Interphotoreceptor Retinoid ... - IOVS

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Fig. 2. Typical competitive binding curve of the enzyme-linked immunosorbent assay (ELISA). Microtitration plates were coated with purified monkey IRBP at a ...
Immunochemical Distribution of Interphotoreceptor Retinoid-Binding Protein in Selected Species Barbara Wiggerr, Ling Lee, Merlyn Rodrigues, Helen Hess, T. Michael Redmond, and Gerald J. Chader An enzyme-linked immunosorbent assay (ELISA) was used to quantitate interphotoreceptor retinoidbinding protein (IRBP) in tissues of monkey and other species using rabbit antiserum against monkey IRBP. A 1:7500 antiserum dilution was found optimal with the linear range extending to 250 jug IRBP/ml. The highest IRBP concentration was found in cannulation fluid of the monkey interphotoreceptor space, although vitreous and aqueous humors also contained IRBP. The presence of IRBP in the vitreous was confirmed by Western blot and 3H-retinoI binding studies. The pineal gland of monkey and rat also was found to contain IRBP, as assessed by ELISA and immunocytochemistry; IRBP was below the limits of detection in turtle and chicken retina. IRBP levels were uniformly low in retinas of human cases with hereditary retinal degeneration, including retinitis pigmentosa (three cases) and choroideremia (two cases). The presence of IRBP in pineal as well as in the vitreous and aqueous humors may indicate a broader role for this putative retinoid-transport protein than previously suspected. Invest Ophthalmol Vis Sci 27:1041-1049, 1986

Interphotoreceptor retinoid-binding protein (IRBP) is a major component of the retinal extracellular matrix (ECM),1"4 and is the only retinoid-binding species in the interphotoreceptor space.2 Although the function of this protein in the retina is yet not clear, many of its characteristics indicate that it could play a role in intercellular retinoid transport, i.e., between the neurosensory retina and pigment epithelium.5 Several years ago in our initial studies on IRBP (then called the "7S protein"), we reported a similar 7S retinoid-binding species in the bovine brain.6 Although not present in liver and other such tissues, this raised the possibility that IRBP might not be unique to the retina, but rather could be more widely distributed in neural tissues. Another puzzling finding in our early studies was the apparent lack of IRBP in some retinas, notably the chicken retina.7 These studies, however, were all performed using sucrose gradient analysis, a technique that measures the ability to bind exogenously added radiolabeled retinoid, rather than measuring the actual IRBP protein concentration. We have recently purified and characterized IRBP from monkey and bovine retina.8 Having the purified protein has now allowed us to reexamine some of these

questions using an antibody assay for the IRBP protein moiety. In the present communication, we describe the development of a specific enzyme-linked immunosorbent assay (ELISA) for IRBP and its use in studying the tissue distribution of IRBP-like immunoreactivity. Confirmatory evidence using immunohistochemistry and Western blotting is also presented. Materials and Methods Sample Preparation Monkey tissues were obtained through the courtesy of the In Vivo Vaccine Test Section, Office of Biologies, FDA. Animals used were Macaca mulatta, and were exsanguinated under deep barbituate anesthesia before enucleation. Mice used were of the pigmented C-57BL-NIH and albino AKR strains; rats of the RCS control strain were obtained from Dr. H. Hess. Treatment of animals adhered to the ARVO Resolution on the Use of Animals in research. Tissues were removed immediately after death, and were frozen on dry ice and stored at — 70°C until use. Cytosol samples of various tissues were obtained by homogenizing the frozen samples in a minimal amount of TRIS buffer (10 raM TRIS, 2 mM EDTA, pH 7.5) followed by centrifugation at 100,000 X g for 1 hr. Monkey IPM was obtained by a previously described cannulation method.2 To obviate any retinal contamination, monkey vitreous was obtained in the following manner. First, whole monkey eyes were frozen in liquid nitrogen. Then, the anterior segment was removed by

From the Laboratory of Retinal Cell and Molecular Biology and the Laboratory of Ocular Pathology, National Eye Institute, National Institutes of Health, Bethesda, Maryland. Submitted for publication: July 9, 1985. Reprint requests: Dr. Barbara Wiggert, Building 6, Room 218, National Eye Institute, National Institutes of Health, Bethesda, MD 20205.

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analyzed in a manner similar to the frozen vitreous. Monkey aqueous was obtained by paracentesis of eyes prior to enucleation. Total protein concentration in the various samples was determined by the dye-binding assay of Bradford9 using a Bio-Rad kit (Bio-Rad Corp., Rockville Center, NY). Human eye-bank eyes were used to obtain human retinas for the purification of IRBP using the same methods as those used to purify monkey and bovine IRBP.8 Human pineal was a normal specimen obtained at autopsy. Enzyme-Linked Immunosorbent Assay (ELISA) Fig. 1. Enzyme-linked immunosorbent assay (ELISA) of purified monkey IRBP. Antiserum was prepared in the rabbit. Anti-serum dilutions depicted are: A = 1:2500; B = 1:5000; C = 1:10,000; D = 1:20,000; E = 1:50,000. F = demonstration of lack of reactivity when bovine serum albumin is used in place of IRBP with an antibody dilution of 1:2500.

circumferential excision at the pars plana, and the central, core portion of the frozen vitreous body was separated. The vitreous core was homogenized and centrifuged at 100,000 X g for 1 hr. The supernatant was then concentrated approximately 30-fold using a Micro-ProDi Con negative pressure microprotein dialysis concentrator (Pierce Chemical Co.; Rockford, IL). In other fresh eyes, after removal of the anterior segment, the vitreous was excised from the vitreous cavity and 0.6 r

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MONKEY Fig. 2. Typical competitive binding curve of the enzyme-linked immunosorbent assay (ELISA). Microtitration plates were coated with purified monkey IRBP at a concentration of 1 jug/ml. Rabbit antiserum to IRBP was used at a dilution of 1:7500.

Primary antiserum against purified monkey IRBP 8 was raised in rabbits. An enzyme-linked immunosorbent assay (ELISA) was performed using this primary antiserum along with anti-rabbit Ig-/?-galactosidaselinked whole antibody (Amersham; Arlington Heights, IL) and following the Amersham protocol for /3-galactosidase-linked reagents. For direct binding assays, microtitration plates were coated with purified monkey IRBP in concentrations ranging from 0.1 to 50 ng/m\ (50 ^1 per well). Bovine serum albumin (Sigma Chemical Co.; St. Louis, MO) was used as a control protein. Non-specific binding sites were blocked with bovine serum albumin (100 n\ per well of 1% bovine serum albumin in phosphate-buffered saline). Dilutions of rabbit anti-monkey IRBP ranging from 1:2500 to 1:50,000 (50 fi\ per well) were used in the assay. In competitive binding assays, microtitration plates were coated with purified monkey IRBP at a concentration of 1 /ug/1 ml (50 (A per well). Twenty-five fxl per well of either unknown sample or a specified amount of purified monkey IRBP plus 25 n\ of rabbit anti-monkey IRBP at a 1:3750 dilution were used. Appropriate controls omitting either the primary or secondary antibodies were included. Electrophoresis: SDS-polyacrylamide gel electrophoresis of purified human, monkey, and bovine IRBP was performed using a Bethesda Research Laboratory vertical slab gel system2 and a 3.5% stacking gel and 15% running gel (15% acrylamide and 0.08% bisacrylamide to obtain a larger pore-size gel). Silver staining was carried out using a Bio-Rad silver-staining kit (Rockville Centre, NY). Western blotting: SDS-polyacrylamide gel electrophoresis was performed as previously described.10 Half of the gel was fixed and stained, and the other half used for electrophoretic transfer to nitro-cellulose paper by the method of Towbin etal 11 using a Bio-Rad apparatus and protocol. A 1:200 dilution of goat antiserum prepared against purified monkey IRBP or a 1:20 dilution of affinity-purified antibody12 (O.D.28o = 0.8) was the

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primary antibody. Horseradish peroxidase conjugated to rabbit anti-goat (F(ab') 2 fragment (Cappel Scientific Division of Cooper Biomedical, Inc.; Malvern, PA) was the second antibody used at a 1:1500 dilution. Immunocytochemistry: Indirect immunofluorescent staining and immunoperoxidase staining with the avidin-biotin-peroxidase complex method were performed as outlined by Hsu et al.13 Primary antiserum prepared in rabbits to purified monkey IRBP was used after affinity purification with glutaraldehyde-crosslinked IRBP-Sepharose immunoabsorbent.12 Antibody absorption was accomplished by adding an excess of purified IRBP antigen to the antibody, incubating at 4°C for 24 hr, and removing the antibody-antigen precipitate by centrifugation. Animals used were: turtles {Chrysemys picta, St. Croix Biol. Supply; Somerset,

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Table 1. Tissue distribution of IRBP in the monkey and rat IRBP concentration (ng/mg soluble protein)

Sample A—Monkey IPM (Cannulation) Retina Cytosol Vitreous Aqueous Pineal Cytosol Cerebral Cortex Cytosol Cornea Cytosol Lens Cytosol Liver Cytosol Testes Cytosol Cerebral-Spinal Fluid B—Rat* Retina-PE Choroid Pineal Harderian Gland Testes Liver C—Mouse* Retina-PE Choroid (C57-BL-NIH) Retina-PE Choroid (AKR)

700 91 233 183

± 160 ± 24 ± 145 ± 89

7 2 None None None None None

Detected Detected Detected Detected Detected

2 ±0.3 1 None Detected None Detected None Detected 10.2 ± 2.4 3.0 ± 0.8

IPM = Soluble proteins of interphotoreceptor matrix.2 Cytosol = 100,000 X g supernatant fraction. * given as "units" of monkey IRBP immunoreactivity.

WI); chickens {Gallus domesticus, Truslow Farms; Chestertown, MD); rat (RCS tan- and black-hooded normal controls). Samples from these animals were also used for ELISA.

t

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Ion Exchange High-Performance Liquid Chromatography Samples were incubated overnight in the presence of 1 fiM 3H-retinol (14.3 Ci/mmole, New England Nuclear; Boston, MA) before application to a Pharmacia Mono-Q anion exchange column. A gradient elution from 0-0.5 M NaCl in 10 mM TRIS-HC1, pH 7.5, containing 2 mM EDTA was carried out using a Pharmacia FPLC system.

Results

Fig. 3. SDS-polyacrylamide gel electrophoretic pattern of IRBP samples. M: monkey IRBP; H: human IRBP; B: bovine IRBP; S: molecular weight standards. The gel was a 15% acrylamide—0.08% bis-acrylamide gel. IRPB was purified as described by Redmond et al.8 Proteins were visualized using a silver strain.

Rabbit anti-monkey IRBP was used successfully in an enzyme-linked immunosorbent assay (ELISA) to quantitate IRBP. Figure 1 shows a direct binding assay for monkey IRBP using different dilutions of the antiserum. It is clear that, even using a 1:50,000 dilution of primary antiserum, IRBP is still detectable in the ixg range. Controls omitting either the primary or secondary antibodies were completely negative. A typical standard curve obtained in a competitive binding assay is given in Figure 2. Using an antiserum dilution of 1:7500, the linear range extended to 250 ng IRBP/ml.

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Fig. 4. Western blot of monkey vitreous soluble proteins with IRBP antibody. Vitreous was concentrated 30-fold prior to SDS-electrophoresis and transfer to nitrocellulose paper. Goat anti-monkey IRBP was used at a dilution of 1:200. A, Coomassie blue stained gel. S = pre-stained standard proteins. 1 = Monkey vitreous. 2 = Purified monkey retina IRBP. B, Western blot. 1 = Monkey vitreous. 2 = Purified monkey retina IRBP. 0.50M 8

o

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I :JH-RETINOL]

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40 FRACTION NUMBER

Fig. 5. High-performance liquid chromatography (HPLC) of concentrated monkey vitreous proteins after incubation with 1 j*M 3Hretinol overnight at 4°C. Arrow shows position of peak eluting in same position as purified monkey retina IRBP. The nature of the peak at about fraction 16 is yet undefined.

This antibody dilution appeared to be optimal and was routinely used in our assays. Figure 3 shows a silver-stained SDS-polyacrylamide gel on which purified monkey, human, and bovine IRBP were subjected to electrophoresis. By this criterion, there were no contaminating proteins in the human preparation. The human protein had an electrophoretic mobility identical to that of purified monkey IRBP. The reactivity of purified human IRBP with the rabbit anti-monkey IRBP was evaluated, and the standard curves in competitive binding assays using human IRBP proved to be virtually identical to those obtained using the monkey protein (Fig. 2). It was thus possible to use the more easily obtainable monkey protein for ELISA of human tissue samples. Table 1 shows the levels of IRBP-like immunoreactivity in several tissues of the monkey and rat and in the mouse retina-PE complex. Not unexpectedly, monkey IPM obtained by cannulation of the subretinal space according to the method of Pfeffer et al2 exhibited

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a high level of IRBP, with the retinoid-binding protein amounting to about 70% of the soluble proteins in this IPM preparation. Both monkey vitreous and aqueous also contained detectable IRBP-immunoreactivity. When monkey vitreous was concentrated approximately 30-fold and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting, a protein band identical to that obtained with purified monkey IRBP was seen (Fig. 4). In separate experiments, a protein peak which bound added 3H-retinol was observed in concentrated vitreous; it eluted in the same place as purified monkey retina IRBP 8 on ion-exchange HPLC (Fig. 5). A Western blot of monkey aqueous also showed a band of IRBP (Fig. 6, lane B). Monkey pineal gland and, to a lesser extent, monkey cortex exhibited IRBP-immunoreactivity (Table 1). A Western blot of human pineal cytosol demonstrated the presence of an IRBP band in this tissue (Fig. 6, lane A). There was no detectable IRBP in the soluble fraction of monkey cornea, lens, liver, or testes.

—200K

—66.2K



Fig. 7. Immunocytochemical localization of IRBP in the rat pineal gland. Staining was with rabbit anti-monkey IRBP (1:10 dilution) using the avidin-biotin complex (ABC) method 13 with hematoxylin counterstain. a, staining of pineal gland (P). There is no reactivity in the surrounding meninges (arrows), b, At higher magnification, there appears to be predominantly intracellular localization of IRBP in pineal gland (P). Adjacent meninges are indicated (arrows) (X330). c, Control showing absence of staining with anti-serum previously absorbed with IRBP (X220).

—45.0K

Fig. 6. Western blot of soluble proteins of human pineal cytosol (lane A) and monkey aqueous (lane B). Purified monkey IRBP standard is given in lane C. Affinity-purified goat anti-monkey IRBP (O.D.28o = 0.80) was used at a dilution of 1:20. Aqueous was concentrated approximately sixfold prior to SDS-electrophoresis and transfer to nitrocellulose paper.

IRBP was detected in the rat posterior segment by the ELISA technique, albeit at much lower levels than in the monkey (Table 1). Values are given as "units" of immunoreactivity based on the ELISA for the monkey IRBP protein and, due to species variation, etc., may not reflect the actual concentration of IRBP present in the rat tissues. The pineal gland also demonstrated IRBP immunoreactivity, but not the Harderian gland, testes, or liver.

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Table 2. IRBP distribution in regions of the monkey retina IRBP (ng/mg protein) Area

Retina cytosol

PE-choroid cytosol

peripheral equatorial posterior

72 ±34 93 ±22 108 ± 17

19 ± 12 48 ± 3 7 46 ± 3 3

After dissection, IRBP was determined in the cytosol (i.e. 100,000 X g supernatant) fraction of each area.

The mouse posterior segment also demonstrated IRBP, although a 3-fold difference was found between the two strains tested. The pineal gland, which is physically separated from other brain structures in the rat, exhibited IRBP-immunoreactivity as assessed by immunocytochemistry (Fig. 7). Figure 7A demonstrates the staining in pineal compared with little or no staining in surrounding brain tissue. At a higher magnification (Fig. 7B), it appears that IRBP immunoreactivity is mainly intracellular. No staining is observed with antiserum previously absorbed with IRBP (Fig. 7C). Because IRBP is soluble and extracellular, biochemical analyses could underestimate IRBP levels if the neural retina only is dissected and used for analysis. Table 2 shows ELISA results obtained with cytosol samples from different regions of the monkey retina and corresponding areas of PE-choroid. More of the extracellular IRBP remained associated with the retinal samples than with the PE-choroid samples, yet significant amounts of IRBP remain with the PE-choroid. Levels of IRBP is monkey retinal samples were similar to those seen in normal human retinal samples (Table 3). A good deal of variation was present in the distribution of the data in the normal human postmortem samples, even though ELISA replicates usually agreed within about 10%. This variation can probably be ascribed to the fact that IRBP is both soluble and extraTable 3. IRBP in normal and diseased retinas IRBP concentration (ng/mg soluble protein) Choroideremia

Ret. pig. Normal Retinal Area peripheral equatorial posterior macula

50 31 79 18

± 39 ± 9 ± 56 ± 8

90 yr 2

66 yr 2

64 yr

61 yr

19 yr

2

1

3

3 2



1

Values in normal retina are averages of triplicate samples from retinas of 5 individual donors ranging in age from 32-77 yr. Values in affected retinas are from triplicate determinations.

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cellular, as pointed out above, and, thus, upon tissue dissection, can remain either with the retina or PE, or be washed out and lost in the ocularfluids,particularly in the older human eyes. In contrast to this variation, however, a marked and uniform reduction in the amount of IRBP was observed in all the retinal samples from patients with hereditary retinal degeneration. Since we had previously not been able to biochemically detect IRBP in the retinas of some species such as the chicken,7 we felt it of importance to examine retinas of selected species to determine if IRBP could be found immunocytochemically. In the rod-dominant rat retina, staining was readily detected in the interphotoreceptor matrix (Fig. 8A, B). In cone-dominant chicken and cone-exclusive turtle retinas, there was a complete lack of IRBP-immunoreactivity (Fig. 8C-F). In parallel experiments, no IRBP-immunoreactivity was observed using the present ELISA in either chicken or turtle retina cytosol samples. To obviate any artifactual loss of IRBP that could be caused by dissection and separation of retina and PE, the entire retina-PE complex was used for cytosol preparation and ELISA. Discussion The ELISA technique outlined in the present communication appears to be specific for IRBP and sensitive at a level of detection normally seen in such assays. Our results are in agreement with results from several other sources, indicating that IRBP (i.e., its immunoreactivity) is found in highest concentration in the retinal interphotoreceptor matrix, and is not widely distributed in many ocular or other tissues, as are the cellular retinol-binding protein (CRBP)14 or the cellular retinoic acid-binding protein (CRABP).15 Using sucrose gradient analysis, for example, we were unable to detect 3 H-retinol binding to a "7S" IRBP in liver cytosol samples of several species14 as now confirmed by the lack of IRBP-immunoreactivity using the sensitive ELISA technique in monkey and rat liver. We also reported that a "7S retinol-binding species" was detectable in bovine brain using gradient analysis.6 ELISA now confirms that IRBP-like immunoreactivity is present in monkey brain, albeit at a low level. In addition, IRBP-like immunoreactivity is also present in the pineal, the specialized brain region regulating circadian functioning. Our ELISA data (Table 1) are extended using immunohistochemical techniques where IRBP-like immunoreaction products are found in rat (Fig. 2) and in monkey16 pineal. It is not particularly surprising to find IRBP in the pineal, since it is well known that rat pinealocytes in the fetal and early neonatal period have several characteristics in common

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Fig. 8. Histological features and immunocytochemistry of rod-dominant (rat) and cone-dominant (turtle, chick) retina, a, Rat retina showing rod cells with intact inner and outer segments (•). Toluidine blue stain of methacrylate (1 n) section (X540). b, Rat retina showing immunofluorescent staining of interphotoreceptor space (•) with antibodies to IRBP (X500). c, Turtle retina showing cone cells (arrows). (Toluidine blue, X330). d, Turtle retina showing lack of immunofluorescent staining with antibodies to IRBP (X33O). e, Chick retina showing predominantly cone cells. (Toluidine blue, X330). f, Chick retina. Immunofluorescent staining with antiIRBP antibodies shows no reactivity (X35O).

with rod photoreceptor cells.17 Also, the mammalian pineal may have evolved from a sub-mammalian "third eye" and functionally is involved in light-entrainment.18 It is interesting to note that the enzyme rhodopsin kinase, once thought to be specific to retinal rod outer segments, is also found in the pineal gland.19 The presence of IRBP in the vitreous and aqueous humors indicates that this protein, which we know to be synthesized in the retina,1020 is not unique to the retinal interphotoreceptor space. How the IRBP reaches these fluids and the function of IRBP in these

compartments is yet unknown. Although unlikely, synthesis of IRBP by anterior segment tissues has yet to be ruled out. Perhaps IRBP may play a role in retinoid transport in this part of the eye as well, since the protein obtained from the vitreous is capable of binding added 3H-retinol. As a major secreted protein of the retina and a putative product of rod photoreceptor cells,1621 we were interested in the presence or absence of IRBP in inherited retinal degenerations of the human. Because these diseases are often differentially expressed in dif-

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ferent areas of the retina, we felt it also important to determine IRBP in the different areas of the normal (Tables 2, 3) as well as in the affected (Table 3) retinas. Although no clear distribution pattern emerged in the normal retina, IRBP did appear to be somewhat lower in the macular samples of thefivenormal human eyes tested. Johnson et al22 found substantial IRBP immunocytochemical activity in the central region of the developing human retina. This study is not comparable to ours, however, since the macular area is still structurally immature at birth,23 and thus the distribution of IRBP in the adult and neonate may differ. Data in Table 3 verify and extend our previous report demonstrating a lack of the "7S retinol-binding protein" (i.e., IRBP) in the retina of a patient with advanced retinitis pigmentosa24 where photoreceptors were missing, and also those of Bridges et al,25 who also studied an advanced case of recessive RP. More interestingly, ELISA confirms the lack of IRBP in peripheral retinal areas of a 66-year-old patient with dominant RP in which photoreceptors were yet present.26 The present ELISA and histochemical results on retinas of the chicken (cone dominant) and the turtle (cone exclusive) are interesting, since they indicate that these retinas contain little or no antigen that crossreacts with the rabbit antibody against monkey IRBP. This could simply be due to species variation and specificity. However, we believe the situation could be somewhat more complex. Using antisera to bovine and frog IRBP, Fong et al27 have reported IRBP of the usual size to be present in a bird species and a 70,000 molecular weight IRBP in bony fish. Using sucrose gradient analysis, we demonstrated several years ago that 7S IRBP could not be detected in chicken retina, and the only retinol-binding species detected was the 2S cellular retinol-binding protein (CRBP).7 Thus, not only is there not a macromolecule that is antigenically similar to monkey IRBP in chicken retina, but cytosol samples of chicken retina do not exhibit a higher molecular weight retinol-binding species in gradient analysis studies. These combined data raise interesting questions as to the general size and/or antigenic nature of the soluble IRBP found in different species. In summary, the present communication outlines a sensitive and specific assay for primate IRBP and demonstrates that IRBP-like immunoreactivity is present not only in retina but also in vitreous and aqueous humors and in the pineal gland. The high concentration of IRBP in the retinal interphotoreceptor matrix (IPM) and its presence in the pineal bring up several possibilities as to the function of the IRBP protein in these areas. For example, IRBP may yet function as a retinoid-transport protein in the IPM, but, since it con-

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stitutes about 70% of the readily soluble protein in the primate IPM2 and since it is a particularly hydrophobic molecule,8 it may also fulfill adhesive and/or structural functions along with other IPM components. In the pineal, the function of IRBP is similarly unclear, but, if it does act as a retinoid transport vehicle, it may indicate a hitherto unsuspected role for retinoids in pinealocyte functioning. Key words: interphotoreceptor retinoid-binding protein, retina, pineal, vitreous, monkey, brain, vitamin A, antibody, enzyme-linked immunosorbent assay, immunocytochemistry, retinitis pigmentosa

Acknowledgments The authors wish to thank the National RP Foundation Fighting Blindness for aid in obtaining several of the RP eyes used in our study. We also appreciate the cooperation of Mr. John Cogan and his staff in obtaining the monkey tissues.

References 1. Adler A and Martin M: Retinoid-binding proteins in bovine interphotoreceptor matrix. Biochem Biophys Res Commun 108: 1601, 1982. 2. Pfeffer B, Wiggert B, Lee L, Zonnenberg B, Newsome D, and Chader GJ: The presence of a soluble Interphotoreceptor Retinoid-Binding Protein in the retinal interphotoreceptor space. J Cell Physiol 117:333, 1983. 3. Bunt-Milam A and Saari J: Immunocytochemical localization of two retinoid-binding proteins in the vertebrate retina. J Cell Biol 97:703, 1983. 4. Fong S-L, Liou G, Landers R, Alvarez R, and Bridges CD: Purification and characterization of a retinol-binding glycoprotein synthesized and secreted by bovine neural retina. J Biol Chem 259:6534, 1984. 5. Chader GJ, Wiggert B, Lai Y-L, Lee L, and Fletcher R: Interphotoreceptor Retinoid-Binding Protein: a possible role in retinoid transport to the retina. In Progress in Retinal Research, Osborne N and Chader GJ, editors. Oxford, Pergamon Press, 1983, pp. 162-189. 6. Wiggert B, Mizukawa A, Kuwabara T, and Chader GJ: Vitamin A receptors: multiple species in retina and brain and possible compartmentalization in retinal photoreceptors. J Neurochem 30:653, 1978. 7. Wiggert B, Bergsma D, Lewis M, and Chader GJ: Vitamin A receptors: retinol binding in neural retina and pigment epithelium. J Neurochem 29:947, 1977. 8. Redmond T, Wiggert B, Robey F, Nguyen N, Lewis M, Lee L, and Chader G: Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina. Biochemistry 24:787, 1985. 9. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of proteindye binding. Anal Biochem 72:248, 1976. 10. Wiggert B, Lee L, O'Brien PJ, and Chader GJ: Synthesis of Interphotoreceptor Retinoid-Binding Protein (IRBP) by monkey retina in organ culture: effect of monensin. Biochem Biophys ResComm 118:789, 1984.

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11. Towbin H, Staehelin T, and Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350,1979. 12. Kapoor CL and Cho-Chung US: Affinity purification of regulatory subunits of cAMP-dependent protein kinase using crosslinked immunoabsorbent. J Immunol Methods 57:215, 1983. 13. Hsu S-M, Raine L, and Fanger H: Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577, 1981. 14. Wiggert B, Bergsma D, and Chader G: Retinol receptors of the retina and pigment epithelium: further characterization and species variation. Exp Eye Res 22:411, 1976. 15. Wiggert B, Bergsma D, Helmsen R, and Chader G: Vitamin A receptors: retinoic acid binding in ocular tissues. Biochem J 169: 87, 1978. 16. Rodrigues M, Hackett J, Gaskins R, Wiggert B, Lee L, Redmond T, and Chader G: Interphotoreceptor retinoid-binding protein in retinal rod cells and pineal gland. Invest Ophthalmol Vis Sci 27:844, 1986. 17. Zimmerman B and Tso M: Morphologic evidence of photoreceptor differentiation of pinealocytes in the neonatal rat. J Cell Biol 66:60, 1975. 18. Axelrod J: The pineal gland: a neurochemical transducer. Science 184:1341, 1974. 19. Somers R and Klein D: Rhodopsin kinase activity in the mammalian pineal gland and other tissues. Science 226:182, 1984.

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20. Fong S-L, Liou GI, Landers R, Alvarez R, Gonzalez-Fernandez F, Glazebrook P, Lam D, and Bridges CD: Characterization, localization and biosynthesis of an interstitial retinol-binding glycoprotein in the human eye. J Neurochem 42:1667, 1984. 21. Hollyfield J, Fliesler S, Rayborn M, Fong S-L, Landers R, and Bridges CD: Synthesis and secretion of interstitial retinol-binding protein by the human retina. Invest Ophthalmol Vis Sci 26:58, 1985. 22. Johnson AT, Kretzer FL, Hittner H, Glazebrook PA, Bridges CD, and Lam DH: Development of the subretinal space in the preterm human eye: ultrastructure and immunocytochemical studies. J Comp Neurol 233:497, 1985. 23. Hendrickson AE and Yuodelis C: The morphological development of the human fovea. Ophthalmology 91:603, 1984. 24. Bergsma D, Wiggert B, Funahashi M, Kuwabara T, and Chader GJ: Vitamin A receptors in normal and dystrophic human retina. Nature 265:66, 1977. 25. Bridges CD, O'Gorman S, Fong S-L, Alvarez RA, and Berson E: Vitamin A and interstitial retinol-binding protein in an eye with recessive retinitis pigmentosa. Invest Ophthalmol Vis Sci 26:684, 1985. 26. Rodrigues MM, Wiggert B, Hackett J, Lee L, Fletcher RT, and Chader GJ: Dominantly inherited retinitis pigmentosa. Ophthalmology 92:1165, 1985. 27. Fong S-L, Johnson KA, Liou GI, Landers RA, Landry AM Jr, and Bridges CD: Distribution of interstitial retinol-binding proteins in the vertebrates ARVO Abstracts. Invest Ophthalmol Vis Sci26(Suppl):17, 1985.