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mixture is transferred to a scintillation vial containing 4.5 ml scintillation cocktail (0.8% 2,5-diphenyloxazole in toluene). After shaking and phase separation, counts per minute (cpm) are determined. Counts per minute corrected for background and fl-galactosidase activity represent the specific CAT activity. In Fig. 6A the transient transfection results of a TRE-CAT reporter gene with RARfl/e are shown. Induction of CAT activity is dependent on the retinoic acid concentration. In Fig. 6B,C, results obtained with the modified CAT assay 24are shown using the RAER-II hybrid receptor15and an ERE-CAT reporter gene) Retinoic acid is compared to two synthetic retinoids (obtained from M. Dawson), one of which shows a very low activity (Fig. 6B) whereas the other shows activity comparable to that of retinoic acid (Fig. 6C). The altered DNA binding specificity of the RAER-II hybrid receptor allows us to analyze its activity in various cell lines that also contain endogenous RAR. The methods described here are rapid and sensitive procedures for the probing, cloning, and analysis of nuclear retinoic acid receptors and their ligands. The procedures can be easily adapted for the general analysis of other proteins.
[2 7] R a d i o i m m u n o a s s a y s for R e t i n o l - B i n d i n g P r o t e i n , Cellular Retinol-Binding Protein, and Cellular Retinoic Acid-Binding Protein B y W I L L I A M S. B L A N E R
Introduction
Serum retinol-binding protein (RBP) is responsible for the transport of retinol from retinoid stores in the liver to target tissues throughout the body. This protein has been extensively studied since its first isolation in 1968,1 and a great deal is now known about its chemical structure and physiologic properties. 2,3 Much of the quantitative information regarding RBP levels in cells, tissues, and serum in both the normal and diseased states has come from studies employing radioimmunoassay (RIA) techM. Kanai, A. Raz, and D. S. Goodman, J. Clin. Invest. 47, 2504 (1968). z D. S. Goodman, in "The Retinoids" (M. B. Sporn, A. B. Roberts, and D. S. Goodman, eds.), Vol. 2, p. 41. Academic Press, Orlando, Florida, 1984. 3 W. S. Blaner, Endocr. Rev. 10, 308 (1989).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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niques for the measurement of RBP. The first portion of this chapter describes procedures, which have been employed in many studies of RBP distribution and function, for the determination of RBP levels by RIA. Within cells, retinol and retinoic acid are associated with specific intracellular retinoid-binding proteins, namely, cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP). 4 These two immunologically distinct proteins appear to be the most widely distributed of the intracellular retinoid-binding proteins, having been reported to be present in all rat tissues e x a m i n e d ) The greatest sensitivity and specificity for measuring either CRBP or CRABP levels, within tissues or cells, have been obtained through the use of RIA techniques. RIA procedures for the measurement of CRBP and CRABP levels are described in the second part of this chapter. Radioimmunoassay for Retinol-Binding Proteins Radioimmunoassay procedures for the determination of RBP levels provide sensitive, specific, and very reproducible methods for the measurement of RBP in serum, 6,7 tissues,8 and isolated cells.9 The RIA procedure is especially useful if RBP levels from a large number of samples are to be determined. The procedure described below was first reported by Smith et aL to for rat RBP; with appropriate substitutions of antisera, iodinated proteins, and standards, it can be used essentially as described for the measurement of human or rat RBP, transthyretin (TTR), and albumin. Preparation of Antiserum
Purified RBP (human or rat) is required for preparation of monospecific polyvalent anti-RBP antisera. The procedures described in this volume for the purification of serum RBP H provide suitable RBP preparations for this purpose. Anti-RBP antisera can be prepared readily in albino rabbits (2-3 kg b o d y weight). A solution of purified RBP (either human or rat) is dis4 F. Chytil and D. E. Ong, in "The Retinoids" (M. B. Sporn, A. B. Roberts, and D. S. Goodman, eds.), Vol. 2, p. 90. Academic Press, Orlando, Florida, 1984. 5 M. Kato, W. S. Blaner, J. R. Mertz, K. Das, K. Kato, and D. S. Goodman, J. Biol. Chem. 2611,4832 (1985). 6 F. R. Smith, A. Raz, and D. S. Goodman, J. Clin. Invest. 49, 1754 (1971). 7 y. Muto, J. E. Smith, P. O. Milch, and D. S. Goodman, J. Biol. Chem. 247, 2542 (1972). 8 j. E. Smith, Y. Muto, and D. S. Goodman, J. Lipid Res. 16, 318 (1975). 9 j. L. Dixon and D. S. Goodman, J. Cell. Physiol. 130, 7 (1987). t0 j. E. Smith, D. D. Deen, Jr., D. Sklan, and D. S. Goodman, J. LipidRes. 21, 229 (1980). it W. S. Blaner and D. S. Goodman, this volume [19].
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solved in 0.9% NaC1 to a concentration of 1 mg/ml and emulsified in an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, M1). Rabbits are immunized by intradermal injection, into approximately 15 sites on the back, of the RBP emulsion (1 mg RBP/rabbit). One month later, booster injections of RBP (1 mg/ml 0.9% NaCI) emulsified in an equal volume of Freund's incomplete adjuvant are given so that 250/xg of RBP is injected intradermally into approximately 10 sites over the back and 250/xg RBP is injected intramuscularly into the hind legs. The titer of anti-RBP antibodies reaches a maximum 10-14 days after this injection. The animal can be bled at this time to provide useful antiserum for the R1A or, alternatively, can be given a second booster injection of RBP to increase further the titer of anti-RBP antibodies. This second booster injection is given 1 month after the first and is given in a manner identical to the first booster injection. Anti-RBP antibody titer is highest 10-14 days after this second injection. The rabbit anti-RBP antisera prepared in this manner should be distributed in small volumes in a series of plastic or glass vials or tubes and stored at - 7 0 °. Under these conditions, rabbit anti-RBP antiserum has been found to remain stable for over 15 years. lodination o f Retinol-Binding Protein
Pure RBP is iodinated by the lactoperoxidase procedure essentially as described by Miyachi et al. 12For this purpose, a stock solution containing 1 mg bovine milk lactoperoxidase (Sigma, St. Louis, MO) in 1 ml of 0.1 M sodium acetate buffer, pH 5.6, is prepared and stored at - 2 0 ° prior to use. Immediately before use, it is necessary to prepare a 0.002% (v/v) H202 solution by first diluting 0.1 ml of 30% (v/v) H202 in 10 ml deionized water followed by further dilution of 0.1 ml of this newly made dilute H202 solution in 15 ml of deionized water. The iodination of RBP is carried out by adding 10/.d of a stock solution of purified RBP (human or rat) containing 5/zg RBP in water, 25/.d of 0.4 M sodium acetate buffer, pH 5.6, and 1 mCi 125I (New England Nuclear, Boston, MA) to a 10 × 75 mm glass tube and mixing gently. To this mixture, 10 p.l of a lactoperoxidase solution, which is prepared immediately before use by diluting 0.1 ml of the stock lactoperoxidase solution in 10 ml of 0.1 M sodium acetate buffer, pH 5.6, is added, and the tube is again mixed gently. To start the enzymatic reaction, 10/zl of the 0.002% H202 solution is added, and the reaction tube is gently mixed and allowed to incubate at 37° for 30 rain. The 125I-labeled RBP is separated from tz y . Miyachi, J. L. Vaitukatis, E. Nieschlag, and M. B. Lipsett, J. Clin. Endocrinol. Metab. 34, 23 (1972).
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nonprotein-bound lzsI by chromatography on a PD-10 column (Pharmacia LKB Biotechnology, Inc., Piscataway, N J) in a buffer consisting of 70 mM Tris-HCl, pH 8.6, containing 70 mM NaCI and 1% bovine serum albumin (BSA). The ~25I-labeled RBP is stored in 70 mM Tris-HCl buffer, pH 8.6, containing 70 mM NaCI and 5% BSA at - 2 0 °. Under these storage conditions, the 125I-labeled RBP can be used in the RIA for up to 2 months.
Radioimmunoassay Procedure Reagents. For the assay of RBP, five reagents are needed. These are RIA buffer, diluted 125I-labeled RBP, diluted anti-RBP antiserum, a stock polyethylene glycol solution, and precipitation buffer. RIA buffer: 50 mM Tris-HCl, pH 8.6, containing 1% BSA. This buffer should be filtered through Whatman # 4 filter paper (Whatman International Ltd., Maidstone, UK), prior to use, to remove flocculent material. This solution can be stored at 4° for up to 2 weeks. Diluted lzSI-labeled RBP: Within 2 hr of use, the lZSI-labeled RBP should be diluted in the RIA buffer so that approximately 20,000 counts per minute (cpm) of 125Iare present in 200/~l of RIA buffer. Diluted anti-RBP antiserum: Within 2 hr of use, the anti-RBP antiserum should be diluted so that the final concentration of antiserum present in the RIA will be sufficient to bind 60% of the ~zSI-labeled RBP present in the assay tube in the absence of added unlabeled RBP. [For rabbit anti-RBP antiserum collected after two booster injections, this will normally be a 1/10,000-1/20,000 (v/v) final (in the assay tube) dilution of the antiserum.] Polyethylene glycol stock solution: 260 g polyethylene glycol 8000 (average molecular weight 7000-9000) (J. T. Baker Chemical Co., Phillipsburg, N J) is added to 3 l0 ml deionized water and allowed to dissolve overnight at room temperature to give a 50% (w/v) solution. This solution can be stored for several months at 4° . Precipitation solution: Prepared immediately before needed, 0.2 g bovine y-globulin (Schwarz/Mann Biotech, Cleveland, OH) is dissolved in 12 ml deionized water and added to 56 ml RIA buffer. To this y-globulin-containing RIA buffer is added 32 ml of the polyethylene glycol stock solution. The solution should become lactescent on addition of the polyethylene glycol to the y-globulin-containing RIA buffer. As described, the procedure provides 100 ml of precipitation solution; however, the procedure can be scaled up as needed.
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RECEPTORS, TRANSPORT, AND BINDING PROTEINS
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Procedure. The reagents for the RBP assay are added to 12 × 75 mm polystyrene conical tubes in the following order: 1. 100/xl of sample to be assayed (either a standard solution of RBP or a diluted serum sample or diluted tissue homogenate) 2. 200/xl of the diluted 125I-labeled RBP (-20,000 cpm) 3. 200/zl of the diluted anti-RBP antiserum [final antiserum dilution 1/10,000-1/20,000 (v/v)] The assay tubes are mixed well with a vortex mixer and incubated at 4 ° for 3 days. For precipitation of the immunoglobulins, 1.5 ml of freshly prepared ice-cold precipitation buffer is added to each assay tube and thoroughly mixed with a vortex mixer. The assay tubes are centrifuged at 4000 g for 30 min at 4 °. The resulting clear supernatant is removed by aspiration, and the pelleted ~25Ipresent in each tube is determined. Standard solutions containing known quantities of purified RBP are prepared in RIA buffer. The concentration of the pure RBP is determined spectrophotometrically using an E l~m of 19.4. ~For the assay of serum (or plasma) RBP levels, samples are diluted in RIA buffer. The diluted sera can be stored for at least 1 week at - 2 0 °. Tissue levels of RBP can be measured in homogenates prepared from the tissues. Tissues are homogenized in 4 volumes (v/w) of RIA buffer containing 1% Triton X-100 with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) at setting 5 for 30 sec. The homogenate is centrifuged at 8000 g for 30 min at 4°, and the resulting supernatants are used directly for the radioimmunoassay of RBP. Similarly, levels of RBP in isolated cells can be obtained by homogenizing a cell pellet of approximately 10 7 cells in 1.0 ml RIA buffer containing 1% Triton X-100 with a Polytron homogenizer, at setting 5 for 30 sec. The cell homogenates are centrifuged at 8000 g for 30 min at 4 °, and the supernatants are removed for RBP assay. If further dilution of the tissue or cell supernatants is necessary, the dilutions should be made in RIA buffer containing 1% Triton X-100. Discussion The procedure described above provides a highly reproducible method for measuring RBP levels in serum (or plasma), tissues, and cells. For the measurement of human serum RBP levels the within- and between-assay coefficients of variation are, respectively, 6.8 and 8.4%. 13 The assay for RBP is very sensitive and is able to measure accurately RBP levels as low as 3 ng per tube. Figure 1 shows a standard displacement curve for purl13 G. D. Friedman, W. S. Blaner, D. S. Goodman, J. H. Vogelman, J. L. Brind, R. Hoover, B. H. Fireman, and N. Orentreich, Am. J. Epidemiol. 123, 781 (1986).
[27]
RADIOIMMUNOASSAYS voR RBP, CRBP, AND CRABP
275
I00 o
B0
z~ 2O
0
o.~
,io
J
2~ 21o 4~o o~o ,~o 32.0 d.o 128 ' no RBPIASSAY TUBE(=
I
I
I
I
=)
I
o.o,8 0.039 ooro o.l~ o.31 o.~z I.~
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p.I SERUM/ASSAY TUBE (o) FIG. 1. Displacement of antibody-bound ~25I-labeled RBP by pure human RBP (Q) and by serial dilutions of an individual human serum specimen ((3).
fled human RBP and diluted human serum. The useful range of this assay is between approximately 3 and 50 ng RBP per assay tube. It has not been possible to use polyvalent rabbit anti-rat RBP antiserum to measure human RBP levels, nor has it been possible to use polyvalent rabbit anti-human RBP antiserum to measure rat RBP levels. Antiserum directed against rat RBP, however, has been used successfully to measure relative levels of RBP in mouse plasma. 14 As a word of caution, the most critical factor for ensuring success in carrying out the RBP RIA is maintaining the pH of all solutions at 8.6. If this pH is not maintained, the immunoglobulins are not properly precipitated. This can be most directly seen with the precipitation buffer, which will not become lactescent on addition of the polyethylene glycol solution. It is now possible to purchase purified human RBP from Calbiochem Corp. (San Diego, CA) and Chemicon International, Inc. (Temecula, CA) and polyclonal anti-RBP from Chemicon and Accurate Chemical and Sci~4A. Brouwer, W. S. Blaner, A. Kukler, and K. J. van den Berg, Chem.-Biol. Interact. 68, 203 (1988).
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RECEPTORS, TRANSPORT, AND BINDING PROTEINS
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entific Corp. (Westbury, NY). Although it seems likely that these commercially available reagents will be useful for the RIA of RBP, this has not been reported in the literature. Radioimmunoassays for Cellular Retinoid-Binding Protein and Cellular Retinoic Acid-Binding Protein Radioimmunoassays for CRBP have been reported by four laboratories, 5,15-18 whereas two radioimmunoassays for CRABP 5,~6 have been described in the literature. Unfortunately, the tissue CRBP and/or CRABP levels reported by different laboratories have not always been in agreement. The basis for the differences in the levels observed between laboratories is not fully clear; however, methodological differences in how the antibodies against CRBP or CRABP were produced and the presence or absence of detergents in the radioimmunoassays may account for some of the lack of agreement between laboratories. The procedures described below have been employed in a variety of studies exploring the cellular and tissue distributions of CRBP and C R A B P ) ,~9-22 The procedures for the two RIAs are identical except for the concentration of antibodies used in each assay.
Preparation of Antisera CRBP and CRABP must be homogeneously pure for the preparation of polyvalent antisera according to the procedures described below. For the procedures described below, CRBP and CRABP were purified from rat testis homogenates by protocols described elsewhere23; however, the purification procedures described previously 24 provide comparably purified CRBP and CRABP for use as immunogens. ~5 N. Adachi, J. E. Smith, and D. S. Goodman, J. Biol. Chem. 256, 9471 (1981). 16 D. E. Ong and F. Chytil, J. Biol. Chem. 257, 13385 (1982). ~7G. Fex and G. Johannesson, Cancer Res. 44, 3029 (1984). 18 U. Eriksson, K. Das, C. Busch, H. Nordlinder, L. Rask, J. Sundelin, J. Sallstrrm, and P. A. Peterson, J. Biol. Chem. 259~ 13464 (1984). ~9W. S. Blaner, K. Das, J. R. Mertz, S. R. Das, and D. S. Goodman, J. LipidRes. 27, 1084 (1986). 20 W. S. Blaner, H. J. F. Hendriks, A. Brouwer, A. M. de Leeuw, D. L, Knook, and D. S. Goodman, J. Lipid Res. 26, 1241 (1985). 2x M. Kato, K. Kato, W. S. Blaner, B. S. Chertow, and D. S. Goodman, Proc. Natl. Acad. Sci. U.S.A. 82, 2488 (1985). z2 W. S. Blaner, M. Galdieri, and D. S. Goodman, Biol. Reprod. 36, 130 (1987). 23 M. Kato, K. Kato, and D. S. Goodman, J. Cell Biol. 98, 1696 0984). 24 F. Chytil and D. E. Ong, in "The Retinoids" (M. B. Sporn, A. B. Roberts, and D. S. Goodman, eds.), Vol. 2, p. 89. Academic Press, Orlando, Florida, 1984.
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Preparation of Anti-CRBP. Antiserum against CRBP is prepared by immunizing a female white turkey (weighing - 8 kg) with 1 ml of a solution of rat testis CRBP (1 mg/ml) in 0.9% NaCI emulsified with an equal volume of Freund's complete adjuvant (Difco). The immunizations are given intramuscularly into multiple sites in the breast muscle. One month after the initial injections, a series of multiple small booster injections consisting of a total of 0.2 mg CRBP emulsified with Freund's incomplete adjuvant is given at the same location as the original injections. A second series of multiple small booster injections, identical in content and site to the first series of booster injections, is given 1 month after the initial booster injections. Antibody titers for CRBP reach a maximum 6 to 9 days after the second booster injection, and blood is collected from the animal during this time interval for use in the RIA for CRBP. The IgG fraction of the turkey plasma is obtained by column chromatography using the procedure of Saif and Dohms. z5 Purified monospecific IgG is then obtained by immunosorbent affinity chromatography of the whole turkey IgG fraction with CRBP linked to Sepharose 4B (Pharmacia LKB Biotechnology). 23 For use in the CRBP RIA, 1 mg of purified turkey IgG against CRBP is mixed with 49 mg preimmune turkey IgG in 5.0 ml of 0.9% NaC1 and stored at - 8 0 ° until use. Preparation of Anti-CRABP. For use as an immunogen, purified CRABP is conjugated with poly(L-lysine). The conjugation is accomplished by adding 300/xg of succinylated poly(L-lysine) (Sigma) and 500 /xg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma) to a solution of CRABP (1 mg/2.7 ml water) and allowing the reaction to proceed overnight at room temperature. The reaction mixture is dialyzed overnight against 0.9% NaCI prior to use in immunization. A portion of the solution of CRABP conjugated to the poly(L-lysine) equivalent to 340 /~g CRABP is emulsified with 1.2 ml of Freund's complete adjuvant and with 120/xg Mycobacterium butyricum (Difco) and injected intramuscularly into multiple sites of the breast of a female white turkey (weighing - 8 kg). An intramuscular booster injection consisting of a portion of the solution of CRABP conjugated to poly(L-lysine) (containing 200/~g CRABP) emulsified with 0.6 ml of Freund's incomplete adjuvant is given 1 month after the initial immunization. The titer of antibodies against CRABP reaches a maximum 5 to 7 days after the booster injection, and blood should be taken at this time for use in the CRABP RIA. Purified monospecific IgG against CRABP is obtained by first purifying whole turkey IgG 25 and subjecting this fraction to immunosorbent affinity chromatography on CRABP linked to Sepharose 4 B . 23 One milli25 y . M. Saif and J. E. Dohms, Avian Dis. 20, 79 (1976).
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RECEPTORS, TRANSPORT, AND BINDING PROTEINS
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gram of purified turkey antibodies against CRABP is mixed with 49 mg preimmune turkey IgG in 5.0 ml of 0.9% NaCl and stored at - 8 0 ° for use in the RIA for CRABP. Preparation of Anti-Turkey IgG. Antibodies for the immunoprecipitation of turkey IgG in the RIAs are prepared in albino rabbits ( - 3 kg body weight). Turkey IgG purified from preimmune turkey plasma 25 is dissolved in 0.9% NaCl, to a concentration of 1 mg/ml, and emulsified in an equal volume of Freund's complete adjuvant. The turkey IgG emulsion is injected intradermally (1 mg/rabbit) over the shoulders of the rabbit in about 15 separate sites. After 1 month, intradermal booster injections of turkey IgG (0.7 mg/rabbit), emulsified in Freund's incomplete adjuvant, are given in multiple sites over the midback. The highest titer of antibodies against turkey IgG is present 6 weeks after the booster injections. The whole antiserum taken at this time is stored at - 8 0 ° and used without further purification in the RIAs for CRBP and CRABP.
Iodination of CRBP and CRABP Both CRBP and CRABP are acylated with ~25I-labeled Bolton-Hunter reagent (4000 Ci/mmol) (New England Nuclear) according to the manufacturer's instructions. Briefly, 10/.~g of CRBP or CRABP, in 5/zl of 0.1 M borate buffer, pH 8.5, is added to the dried iodinated ester, and the reaction mixture is continuously mixed for 20 min at 0° and then allowed to stand for 40 min at 0 °. Following this incubation, the reaction is quenched by adding 100/xl of 0.5 M glycine in 0. I M borate buffer, pH 8.5, and incubating for 60 min at 0°. The acylated proteins are separated from nonprotein-bound ~2sI by chromatography on PD-10 columns (Pharmacia LKB Biotechnology) using 10 m M sodium phosphate, pH 7.2, containing 150 m M NaCI and I% BSA. Following this procedure, the specific activities of the resulting acylated CRBP and CRABP preparations normally are in the range of 20 to 40 mCi/mg.
Radioimmunoassay Procedures The radioimmunoassay procedures for CRBP and CRABP are identical. All reagents are dissolved or diluted in a buffer (RIA buffer) consisting of 50 mM imidazole-HCl, pH 7.4, containing 150 m M NaCI, 0.03% BSA, 0.1% thimerosal (Sigma), 0.01% leupeptin (Peninsula Laboratories, San Carlos, CA), and 1% Triton X-100. All procedures are carried out on ice or at 4°. Reagents are added to polystyrene conical tubes (12 x 75 mm) and incubated in the following sequence: 1. 100/.d of sample to be assayed (either a standard solution of CRBP or CRABP, or a diluted tissue or cellular cytosol)
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2. 50/zl of diluted anti-CRBP IgG solution (containing 0.04/zg purified specific IgG and 1.96/zg of preimmune IgG) or diluted anti-CRABP IgG solution (containing 0.02/zg of purified specific IgG and 0.98/xg of preimmune IgG) 3. 300/xl of RIA buffer; incubate for 3-4 hr 4. 50 /A of 125I-labeled CRBP or 125I-labeled CRABP (-20,000 cpm ~25I); incubate 20-24 hr at 4 ° with gentle agitation 5. 25/zl of rabbit antisera against turkey IgG; incubate 12-18 hr at 4 ° with gentle agitation. Samples are centrifuged at 4000 g for 30 min at 4 °, the supernatants are removed by aspiration, and the precipitates are assayed for ~25I. The standard solutions of CRBP or CRABP are prepared from purified CRBP or CRABP diluted in RIA buffer. The concentrations of pure CRBP and CRABP are estimated spectrophotometrically using ~_ll - - E, '~ ~l cm of 14. 26 For both the CRBP RIA and the CRABP RIA, the working ranges of the assays are between 1 and 16 ng of CRBP or CRABP per assay tube. Tissue cytosol preparations are prepared from perfused (with ice-cold 0.9% NaCI) freshly dissected tissues. The dissected tissues are minced, and 500 mg of the tissue mince is added to 3.0 mi RIA buffer and homogenized with a Polytron homogenizer (Brinkmann Instruments) at setting 5 for 30 sec. The tissue homogenates are centrifuged at 105,000 g for 1 hr, and the resulting clear supernatant is diluted appropriately with RIA buffer for assay. Cell pellets containing approximately 10 7 cells are homogenized in 1.0 ml RIA buffer with a Polytron homogenizer on setting 5 for 30 sec. Cytosols from the cell homogenates are prepared by centrifugation of the homogenates at 105,000 g for 1 hr.
Discussion
The procedures described above provide sensitive, specific, and reproducible methods for measuring CRBP and CRABP levels in tissues or isolated cells. Levels of CRBP and CRABP determined with these two RIA procedures, for some tissues and cell types, are given in Table I. For the CRBP RIA, the values for the within-assay and for the between-assay variability are estimated to be 2.7 and 4.8%, respectively. 5 The lower detection limit of the CRBP RIA is 1 ng CRBP/assay tube; hence, it is possible to measure accurately tissue levels as low as 0.07/zg CRBP/g tissue or cellular levels as low as 1 ng CRBP/106 cells. The within-assay and between-assay variabilities for the RIA of CRABP are estimated to be 26 A. C. Ross, Y. I. Takahashi, and D. S. Goodman, J. Biol. Chem. 253, 6591 (1978).
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RECEPTORS, TRANSPORT, AND BINDING PROTEINS
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TABLE I LEVELS OF CRBP AND CRABP IN VARIOUS RAT TISSUES AND CELLS
Tissue or cell
CRBP~
Liverb 40.0 ± 9.7 Small intestineb 10.3 -+ 1.4 Brainb 4.4 ± 0.5 Skinb 3.5 ± 0.6 Fat b 1.2 -+ 0.3 Testis b 17.8 ± 4.8 Seminal vesicle b 2.9 ± 0.7 Hepatic parenchymal cells c 470 ± 238 Hepatic stellate cells c 236 ± 89
CRABP° 1.1 -+ 0.2 0.8 -+ 0.2 1.2 ± 0.3 12.6 ± 2.3 0.2 - 0.1 9.5 -+ 1.1 30.4 ± 7.9 5.6 -+ 3.9 8.7 ± 5.7
Tissue levels of CRBP and CRABP are given as micrograms CRBP or CRABP per gram wet weight of the tissue. Cellular levels of CRBP and CRABP are given as nanograms CRBP or CRABP per 106 cells. All values are given as the mean --+ 1 S.D. Levels of CRBP and CRABP are from Ref. 5. Levels of CRBP and CRABP are from Ref. 20. When these levels are expressed on the basis of cellular protein, the hepatic parenchymal cell levels of CRBP and CRABP are 243 ± 124 ng CRBP/mg protein and 2.9 --- 3.3 ng CRABP/mg protein; hepatic stellate cell levels of CRBP and CRABP are 1256 --- 477 ng CRBP/mg protein and 44 --- 30 ng CRABP/mg protein.
4.1 a n d 5.5%, r e s p e c t i v e l y ) L o w e r d e t e c t i o n limits for the C R A B P R I A are 0 . 0 7 / ~ g C R A B P / g t i s s u e a n d 1 ng C R A B P / 1 0 6 cells. I n c a r r y i n g o u t the a s s a y s for C R B P a n d C R A B P , it is i m p o r t a n t to a d h e r e to the p r o t o c o l s d e s c r i b e d a b o v e . T h e t i m e s a n d t e m p e r a t u r e s o f i n c u b a t i o n s are i m p o r t a n t for the s e n s i t i v i t i e s (affecting the w o r k i n g r a n g e s ) o f e a c h o f the R I A s . T h e p r e p a r a t i o n s o f 125I-labeled C R B P o r 125Il a b e l e d C R A B P s h o u l d h a v e specific a c t i v i t i e s b e t w e e n 20 a n d 40 m C i / m m o l . I f C R B P o r C R A P B are e i t h e r m o r e o r less h e a v i l y l a b e l e d with 1251, the s t a n d a r d d i s p l a c e m e n t c u r v e s are f o u n d to shift to l o w e r s e n s i t i v ity. T h e 125I-labeled C R B P a n d 125I-labeled C R A B P c a n be s t o r e d in 1% B S A at - 2 0 ° for u p to 1 m o n t h . T i s s u e o r cell c y t o s o l p r e p a r a t i o n s c a n b e s t o r e d o v e r n i g h t at 4 ° o r for u p to 14 d a y s at - 8 0 °. S t o r a g e o f c y t o s o l p r e p a r a t i o n s for l o n g e r t h a n 14 d a y s at - 8 0 ° r e s u l t s in a d e c r e a s e in the i m m u n o r e a c t i v e C R B P a n d C R A B P c o n t e n t s o f the c y t o s o l p r e p a r a t i o n s . It is n e c e s s a r y to titer e a c h n e w b a t c h o f s e c o n d a n t i b o d y ( r a b b i t anti-
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281
turkey IgG) employed in the assays, to ensure that this antibody is not present in limiting concentrations. For eight different rabbit anti-turkey IgG antisera examined by this author, 25 p,l of each antisera provided a large excess of anti-turkey IgG antisera; however, this need not always be true. Acknowledgments The author would like to thank Drs. DeWitt S. Goodman and John E. Smith for their help and support. This work was supported by National Institutes of Health Grants DK 05968 and HL 21006.
[28] X - R a y C r y s t a l l o g r a p h i c S t u d i e s on Retinol-Binding Proteins
By MARCIA NEWCOMER and T. ALWYN JONES Introduction Structural information for proteins which interact with retinoids, in their transport or metabolism, or which mediate retinoid function(s) is critical to our understanding of the role(s) of natural or synthetic retinoids in physiology and medicine. X-Ray crystallographic studies of this class of proteins can provide the details we need to understand retinoid recognition at the molecular level. The steps involved in a structural determination can be briefly summarized as follows. (1) The protein must be isolated in relatively large quantity (> 10 mg) with a very high degree of purity. (2) Protein crystals suitable for X-ray diffraction data collection are prepared. (3) X-Ray diffraction data are collected. (4) The "phase problem" is solved by an appropriate method. (5) An electron density map is calculated. (6) A model for the protein structure is built into the electron density. (7) The model is refined. Once a high-resolution structure is determined, the structures of the protein complexed with ligand or ligand analogs are relatively straightforward to determine. The three-dimensional structure of the retinol-binding protein (RBP) from human serum has been determined by the process briefly outlined above. 1 For information with respect to the standard methods in protein structure determination employed, the reader is referred to Volumes 114 i M. E. Newcomer, T. A. Jones, J. ~quist, J. Sundelin, U. Eriksson, L. Rask, and P. A. Peterson, EMBO J. 3, 1451 (1984).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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mixture is transferred to a scintillation vial containing 4.5 ml scintillation cocktail (0.8% 2,5-diphenyloxazole in toluene). After shaking and phase separation, counts per minute (cpm) are determined. Counts per minute corrected for background and fl-galactosidase activity represent the specific CAT activity. In Fig. 6A the transient transfection results of a TRE-CAT reporter gene with RARfl/e are shown. Induction of CAT activity is dependent on the retinoic acid concentration. In Fig. 6B,C, results obtained with the modified CAT assay 24are shown using the RAER-II hybrid receptor15and an ERE-CAT reporter gene) Retinoic acid is compared to two synthetic retinoids (obtained from M. Dawson), one of which shows a very low activity (Fig. 6B) whereas the other shows activity comparable to that of retinoic acid (Fig. 6C). The altered DNA binding specificity of the RAER-II hybrid receptor allows us to analyze its activity in various cell lines that also contain endogenous RAR. The methods described here are rapid and sensitive procedures for the probing, cloning, and analysis of nuclear retinoic acid receptors and their ligands. The procedures can be easily adapted for the general analysis of other proteins.
[2 7] R a d i o i m m u n o a s s a y s for R e t i n o l - B i n d i n g P r o t e i n , Cellular Retinol-Binding Protein, and Cellular Retinoic Acid-Binding Protein B y W I L L I A M S. B L A N E R
Introduction
Serum retinol-binding protein (RBP) is responsible for the transport of retinol from retinoid stores in the liver to target tissues throughout the body. This protein has been extensively studied since its first isolation in 1968,1 and a great deal is now known about its chemical structure and physiologic properties. 2,3 Much of the quantitative information regarding RBP levels in cells, tissues, and serum in both the normal and diseased states has come from studies employing radioimmunoassay (RIA) techM. Kanai, A. Raz, and D. S. Goodman, J. Clin. Invest. 47, 2504 (1968). z D. S. Goodman, in "The Retinoids" (M. B. Sporn, A. B. Roberts, and D. S. Goodman, eds.), Vol. 2, p. 41. Academic Press, Orlando, Florida, 1984. 3 W. S. Blaner, Endocr. Rev. 10, 308 (1989).
METHODS IN ENZYMOLOGY, VOL. 189
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niques for the measurement of RBP. The first portion of this chapter describes procedures, which have been employed in many studies of RBP distribution and function, for the determination of RBP levels by RIA. Within cells, retinol and retinoic acid are associated with specific intracellular retinoid-binding proteins, namely, cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP). 4 These two immunologically distinct proteins appear to be the most widely distributed of the intracellular retinoid-binding proteins, having been reported to be present in all rat tissues e x a m i n e d ) The greatest sensitivity and specificity for measuring either CRBP or CRABP levels, within tissues or cells, have been obtained through the use of RIA techniques. RIA procedures for the measurement of CRBP and CRABP levels are described in the second part of this chapter. Radioimmunoassay for Retinol-Binding Proteins Radioimmunoassay procedures for the determination of RBP levels provide sensitive, specific, and very reproducible methods for the measurement of RBP in serum, 6,7 tissues,8 and isolated cells.9 The RIA procedure is especially useful if RBP levels from a large number of samples are to be determined. The procedure described below was first reported by Smith et aL to for rat RBP; with appropriate substitutions of antisera, iodinated proteins, and standards, it can be used essentially as described for the measurement of human or rat RBP, transthyretin (TTR), and albumin. Preparation of Antiserum
Purified RBP (human or rat) is required for preparation of monospecific polyvalent anti-RBP antisera. The procedures described in this volume for the purification of serum RBP H provide suitable RBP preparations for this purpose. Anti-RBP antisera can be prepared readily in albino rabbits (2-3 kg b o d y weight). A solution of purified RBP (either human or rat) is dis4 F. Chytil and D. E. Ong, in "The Retinoids" (M. B. Sporn, A. B. Roberts, and D. S. Goodman, eds.), Vol. 2, p. 90. Academic Press, Orlando, Florida, 1984. 5 M. Kato, W. S. Blaner, J. R. Mertz, K. Das, K. Kato, and D. S. Goodman, J. Biol. Chem. 2611,4832 (1985). 6 F. R. Smith, A. Raz, and D. S. Goodman, J. Clin. Invest. 49, 1754 (1971). 7 y. Muto, J. E. Smith, P. O. Milch, and D. S. Goodman, J. Biol. Chem. 247, 2542 (1972). 8 j. E. Smith, Y. Muto, and D. S. Goodman, J. Lipid Res. 16, 318 (1975). 9 j. L. Dixon and D. S. Goodman, J. Cell. Physiol. 130, 7 (1987). t0 j. E. Smith, D. D. Deen, Jr., D. Sklan, and D. S. Goodman, J. LipidRes. 21, 229 (1980). it W. S. Blaner and D. S. Goodman, this volume [19].
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solved in 0.9% NaC1 to a concentration of 1 mg/ml and emulsified in an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, M1). Rabbits are immunized by intradermal injection, into approximately 15 sites on the back, of the RBP emulsion (1 mg RBP/rabbit). One month later, booster injections of RBP (1 mg/ml 0.9% NaCI) emulsified in an equal volume of Freund's incomplete adjuvant are given so that 250/xg of RBP is injected intradermally into approximately 10 sites over the back and 250/xg RBP is injected intramuscularly into the hind legs. The titer of anti-RBP antibodies reaches a maximum 10-14 days after this injection. The animal can be bled at this time to provide useful antiserum for the R1A or, alternatively, can be given a second booster injection of RBP to increase further the titer of anti-RBP antibodies. This second booster injection is given 1 month after the first and is given in a manner identical to the first booster injection. Anti-RBP antibody titer is highest 10-14 days after this second injection. The rabbit anti-RBP antisera prepared in this manner should be distributed in small volumes in a series of plastic or glass vials or tubes and stored at - 7 0 °. Under these conditions, rabbit anti-RBP antiserum has been found to remain stable for over 15 years. lodination o f Retinol-Binding Protein
Pure RBP is iodinated by the lactoperoxidase procedure essentially as described by Miyachi et al. 12For this purpose, a stock solution containing 1 mg bovine milk lactoperoxidase (Sigma, St. Louis, MO) in 1 ml of 0.1 M sodium acetate buffer, pH 5.6, is prepared and stored at - 2 0 ° prior to use. Immediately before use, it is necessary to prepare a 0.002% (v/v) H202 solution by first diluting 0.1 ml of 30% (v/v) H202 in 10 ml deionized water followed by further dilution of 0.1 ml of this newly made dilute H202 solution in 15 ml of deionized water. The iodination of RBP is carried out by adding 10/.d of a stock solution of purified RBP (human or rat) containing 5/zg RBP in water, 25/.d of 0.4 M sodium acetate buffer, pH 5.6, and 1 mCi 125I (New England Nuclear, Boston, MA) to a 10 × 75 mm glass tube and mixing gently. To this mixture, 10 p.l of a lactoperoxidase solution, which is prepared immediately before use by diluting 0.1 ml of the stock lactoperoxidase solution in 10 ml of 0.1 M sodium acetate buffer, pH 5.6, is added, and the tube is again mixed gently. To start the enzymatic reaction, 10/zl of the 0.002% H202 solution is added, and the reaction tube is gently mixed and allowed to incubate at 37° for 30 rain. The 125I-labeled RBP is separated from tz y . Miyachi, J. L. Vaitukatis, E. Nieschlag, and M. B. Lipsett, J. Clin. Endocrinol. Metab. 34, 23 (1972).
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nonprotein-bound lzsI by chromatography on a PD-10 column (Pharmacia LKB Biotechnology, Inc., Piscataway, N J) in a buffer consisting of 70 mM Tris-HCl, pH 8.6, containing 70 mM NaCI and 1% bovine serum albumin (BSA). The ~25I-labeled RBP is stored in 70 mM Tris-HCl buffer, pH 8.6, containing 70 mM NaCI and 5% BSA at - 2 0 °. Under these storage conditions, the 125I-labeled RBP can be used in the RIA for up to 2 months.
Radioimmunoassay Procedure Reagents. For the assay of RBP, five reagents are needed. These are RIA buffer, diluted 125I-labeled RBP, diluted anti-RBP antiserum, a stock polyethylene glycol solution, and precipitation buffer. RIA buffer: 50 mM Tris-HCl, pH 8.6, containing 1% BSA. This buffer should be filtered through Whatman # 4 filter paper (Whatman International Ltd., Maidstone, UK), prior to use, to remove flocculent material. This solution can be stored at 4° for up to 2 weeks. Diluted lzSI-labeled RBP: Within 2 hr of use, the lZSI-labeled RBP should be diluted in the RIA buffer so that approximately 20,000 counts per minute (cpm) of 125Iare present in 200/~l of RIA buffer. Diluted anti-RBP antiserum: Within 2 hr of use, the anti-RBP antiserum should be diluted so that the final concentration of antiserum present in the RIA will be sufficient to bind 60% of the ~zSI-labeled RBP present in the assay tube in the absence of added unlabeled RBP. [For rabbit anti-RBP antiserum collected after two booster injections, this will normally be a 1/10,000-1/20,000 (v/v) final (in the assay tube) dilution of the antiserum.] Polyethylene glycol stock solution: 260 g polyethylene glycol 8000 (average molecular weight 7000-9000) (J. T. Baker Chemical Co., Phillipsburg, N J) is added to 3 l0 ml deionized water and allowed to dissolve overnight at room temperature to give a 50% (w/v) solution. This solution can be stored for several months at 4° . Precipitation solution: Prepared immediately before needed, 0.2 g bovine y-globulin (Schwarz/Mann Biotech, Cleveland, OH) is dissolved in 12 ml deionized water and added to 56 ml RIA buffer. To this y-globulin-containing RIA buffer is added 32 ml of the polyethylene glycol stock solution. The solution should become lactescent on addition of the polyethylene glycol to the y-globulin-containing RIA buffer. As described, the procedure provides 100 ml of precipitation solution; however, the procedure can be scaled up as needed.
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Procedure. The reagents for the RBP assay are added to 12 × 75 mm polystyrene conical tubes in the following order: 1. 100/xl of sample to be assayed (either a standard solution of RBP or a diluted serum sample or diluted tissue homogenate) 2. 200/xl of the diluted 125I-labeled RBP (-20,000 cpm) 3. 200/zl of the diluted anti-RBP antiserum [final antiserum dilution 1/10,000-1/20,000 (v/v)] The assay tubes are mixed well with a vortex mixer and incubated at 4 ° for 3 days. For precipitation of the immunoglobulins, 1.5 ml of freshly prepared ice-cold precipitation buffer is added to each assay tube and thoroughly mixed with a vortex mixer. The assay tubes are centrifuged at 4000 g for 30 min at 4 °. The resulting clear supernatant is removed by aspiration, and the pelleted ~25Ipresent in each tube is determined. Standard solutions containing known quantities of purified RBP are prepared in RIA buffer. The concentration of the pure RBP is determined spectrophotometrically using an E l~m of 19.4. ~For the assay of serum (or plasma) RBP levels, samples are diluted in RIA buffer. The diluted sera can be stored for at least 1 week at - 2 0 °. Tissue levels of RBP can be measured in homogenates prepared from the tissues. Tissues are homogenized in 4 volumes (v/w) of RIA buffer containing 1% Triton X-100 with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) at setting 5 for 30 sec. The homogenate is centrifuged at 8000 g for 30 min at 4°, and the resulting supernatants are used directly for the radioimmunoassay of RBP. Similarly, levels of RBP in isolated cells can be obtained by homogenizing a cell pellet of approximately 10 7 cells in 1.0 ml RIA buffer containing 1% Triton X-100 with a Polytron homogenizer, at setting 5 for 30 sec. The cell homogenates are centrifuged at 8000 g for 30 min at 4 °, and the supernatants are removed for RBP assay. If further dilution of the tissue or cell supernatants is necessary, the dilutions should be made in RIA buffer containing 1% Triton X-100. Discussion The procedure described above provides a highly reproducible method for measuring RBP levels in serum (or plasma), tissues, and cells. For the measurement of human serum RBP levels the within- and between-assay coefficients of variation are, respectively, 6.8 and 8.4%. 13 The assay for RBP is very sensitive and is able to measure accurately RBP levels as low as 3 ng per tube. Figure 1 shows a standard displacement curve for purl13 G. D. Friedman, W. S. Blaner, D. S. Goodman, J. H. Vogelman, J. L. Brind, R. Hoover, B. H. Fireman, and N. Orentreich, Am. J. Epidemiol. 123, 781 (1986).
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RADIOIMMUNOASSAYS voR RBP, CRBP, AND CRABP
275
I00 o
B0
z~ 2O
0
o.~
,io
J
2~ 21o 4~o o~o ,~o 32.0 d.o 128 ' no RBPIASSAY TUBE(=
I
I
I
I
=)
I
o.o,8 0.039 ooro o.l~ o.31 o.~z I.~
2'.~
5'.0
p.I SERUM/ASSAY TUBE (o) FIG. 1. Displacement of antibody-bound ~25I-labeled RBP by pure human RBP (Q) and by serial dilutions of an individual human serum specimen ((3).
fled human RBP and diluted human serum. The useful range of this assay is between approximately 3 and 50 ng RBP per assay tube. It has not been possible to use polyvalent rabbit anti-rat RBP antiserum to measure human RBP levels, nor has it been possible to use polyvalent rabbit anti-human RBP antiserum to measure rat RBP levels. Antiserum directed against rat RBP, however, has been used successfully to measure relative levels of RBP in mouse plasma. 14 As a word of caution, the most critical factor for ensuring success in carrying out the RBP RIA is maintaining the pH of all solutions at 8.6. If this pH is not maintained, the immunoglobulins are not properly precipitated. This can be most directly seen with the precipitation buffer, which will not become lactescent on addition of the polyethylene glycol solution. It is now possible to purchase purified human RBP from Calbiochem Corp. (San Diego, CA) and Chemicon International, Inc. (Temecula, CA) and polyclonal anti-RBP from Chemicon and Accurate Chemical and Sci~4A. Brouwer, W. S. Blaner, A. Kukler, and K. J. van den Berg, Chem.-Biol. Interact. 68, 203 (1988).
276
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
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entific Corp. (Westbury, NY). Although it seems likely that these commercially available reagents will be useful for the RIA of RBP, this has not been reported in the literature. Radioimmunoassays for Cellular Retinoid-Binding Protein and Cellular Retinoic Acid-Binding Protein Radioimmunoassays for CRBP have been reported by four laboratories, 5,15-18 whereas two radioimmunoassays for CRABP 5,~6 have been described in the literature. Unfortunately, the tissue CRBP and/or CRABP levels reported by different laboratories have not always been in agreement. The basis for the differences in the levels observed between laboratories is not fully clear; however, methodological differences in how the antibodies against CRBP or CRABP were produced and the presence or absence of detergents in the radioimmunoassays may account for some of the lack of agreement between laboratories. The procedures described below have been employed in a variety of studies exploring the cellular and tissue distributions of CRBP and C R A B P ) ,~9-22 The procedures for the two RIAs are identical except for the concentration of antibodies used in each assay.
Preparation of Antisera CRBP and CRABP must be homogeneously pure for the preparation of polyvalent antisera according to the procedures described below. For the procedures described below, CRBP and CRABP were purified from rat testis homogenates by protocols described elsewhere23; however, the purification procedures described previously 24 provide comparably purified CRBP and CRABP for use as immunogens. ~5 N. Adachi, J. E. Smith, and D. S. Goodman, J. Biol. Chem. 256, 9471 (1981). 16 D. E. Ong and F. Chytil, J. Biol. Chem. 257, 13385 (1982). ~7G. Fex and G. Johannesson, Cancer Res. 44, 3029 (1984). 18 U. Eriksson, K. Das, C. Busch, H. Nordlinder, L. Rask, J. Sundelin, J. Sallstrrm, and P. A. Peterson, J. Biol. Chem. 259~ 13464 (1984). ~9W. S. Blaner, K. Das, J. R. Mertz, S. R. Das, and D. S. Goodman, J. LipidRes. 27, 1084 (1986). 20 W. S. Blaner, H. J. F. Hendriks, A. Brouwer, A. M. de Leeuw, D. L, Knook, and D. S. Goodman, J. Lipid Res. 26, 1241 (1985). 2x M. Kato, K. Kato, W. S. Blaner, B. S. Chertow, and D. S. Goodman, Proc. Natl. Acad. Sci. U.S.A. 82, 2488 (1985). z2 W. S. Blaner, M. Galdieri, and D. S. Goodman, Biol. Reprod. 36, 130 (1987). 23 M. Kato, K. Kato, and D. S. Goodman, J. Cell Biol. 98, 1696 0984). 24 F. Chytil and D. E. Ong, in "The Retinoids" (M. B. Sporn, A. B. Roberts, and D. S. Goodman, eds.), Vol. 2, p. 89. Academic Press, Orlando, Florida, 1984.
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Preparation of Anti-CRBP. Antiserum against CRBP is prepared by immunizing a female white turkey (weighing - 8 kg) with 1 ml of a solution of rat testis CRBP (1 mg/ml) in 0.9% NaCI emulsified with an equal volume of Freund's complete adjuvant (Difco). The immunizations are given intramuscularly into multiple sites in the breast muscle. One month after the initial injections, a series of multiple small booster injections consisting of a total of 0.2 mg CRBP emulsified with Freund's incomplete adjuvant is given at the same location as the original injections. A second series of multiple small booster injections, identical in content and site to the first series of booster injections, is given 1 month after the initial booster injections. Antibody titers for CRBP reach a maximum 6 to 9 days after the second booster injection, and blood is collected from the animal during this time interval for use in the RIA for CRBP. The IgG fraction of the turkey plasma is obtained by column chromatography using the procedure of Saif and Dohms. z5 Purified monospecific IgG is then obtained by immunosorbent affinity chromatography of the whole turkey IgG fraction with CRBP linked to Sepharose 4B (Pharmacia LKB Biotechnology). 23 For use in the CRBP RIA, 1 mg of purified turkey IgG against CRBP is mixed with 49 mg preimmune turkey IgG in 5.0 ml of 0.9% NaC1 and stored at - 8 0 ° until use. Preparation of Anti-CRABP. For use as an immunogen, purified CRABP is conjugated with poly(L-lysine). The conjugation is accomplished by adding 300/xg of succinylated poly(L-lysine) (Sigma) and 500 /xg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma) to a solution of CRABP (1 mg/2.7 ml water) and allowing the reaction to proceed overnight at room temperature. The reaction mixture is dialyzed overnight against 0.9% NaCI prior to use in immunization. A portion of the solution of CRABP conjugated to the poly(L-lysine) equivalent to 340 /~g CRABP is emulsified with 1.2 ml of Freund's complete adjuvant and with 120/xg Mycobacterium butyricum (Difco) and injected intramuscularly into multiple sites of the breast of a female white turkey (weighing - 8 kg). An intramuscular booster injection consisting of a portion of the solution of CRABP conjugated to poly(L-lysine) (containing 200/~g CRABP) emulsified with 0.6 ml of Freund's incomplete adjuvant is given 1 month after the initial immunization. The titer of antibodies against CRABP reaches a maximum 5 to 7 days after the booster injection, and blood should be taken at this time for use in the CRABP RIA. Purified monospecific IgG against CRABP is obtained by first purifying whole turkey IgG 25 and subjecting this fraction to immunosorbent affinity chromatography on CRABP linked to Sepharose 4 B . 23 One milli25 y . M. Saif and J. E. Dohms, Avian Dis. 20, 79 (1976).
278
RECEPTORS, TRANSPORT, AND BINDING PROTEINS
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gram of purified turkey antibodies against CRABP is mixed with 49 mg preimmune turkey IgG in 5.0 ml of 0.9% NaCl and stored at - 8 0 ° for use in the RIA for CRABP. Preparation of Anti-Turkey IgG. Antibodies for the immunoprecipitation of turkey IgG in the RIAs are prepared in albino rabbits ( - 3 kg body weight). Turkey IgG purified from preimmune turkey plasma 25 is dissolved in 0.9% NaCl, to a concentration of 1 mg/ml, and emulsified in an equal volume of Freund's complete adjuvant. The turkey IgG emulsion is injected intradermally (1 mg/rabbit) over the shoulders of the rabbit in about 15 separate sites. After 1 month, intradermal booster injections of turkey IgG (0.7 mg/rabbit), emulsified in Freund's incomplete adjuvant, are given in multiple sites over the midback. The highest titer of antibodies against turkey IgG is present 6 weeks after the booster injections. The whole antiserum taken at this time is stored at - 8 0 ° and used without further purification in the RIAs for CRBP and CRABP.
Iodination of CRBP and CRABP Both CRBP and CRABP are acylated with ~25I-labeled Bolton-Hunter reagent (4000 Ci/mmol) (New England Nuclear) according to the manufacturer's instructions. Briefly, 10/.~g of CRBP or CRABP, in 5/zl of 0.1 M borate buffer, pH 8.5, is added to the dried iodinated ester, and the reaction mixture is continuously mixed for 20 min at 0° and then allowed to stand for 40 min at 0 °. Following this incubation, the reaction is quenched by adding 100/xl of 0.5 M glycine in 0. I M borate buffer, pH 8.5, and incubating for 60 min at 0°. The acylated proteins are separated from nonprotein-bound ~2sI by chromatography on PD-10 columns (Pharmacia LKB Biotechnology) using 10 m M sodium phosphate, pH 7.2, containing 150 m M NaCI and I% BSA. Following this procedure, the specific activities of the resulting acylated CRBP and CRABP preparations normally are in the range of 20 to 40 mCi/mg.
Radioimmunoassay Procedures The radioimmunoassay procedures for CRBP and CRABP are identical. All reagents are dissolved or diluted in a buffer (RIA buffer) consisting of 50 mM imidazole-HCl, pH 7.4, containing 150 m M NaCI, 0.03% BSA, 0.1% thimerosal (Sigma), 0.01% leupeptin (Peninsula Laboratories, San Carlos, CA), and 1% Triton X-100. All procedures are carried out on ice or at 4°. Reagents are added to polystyrene conical tubes (12 x 75 mm) and incubated in the following sequence: 1. 100/.d of sample to be assayed (either a standard solution of CRBP or CRABP, or a diluted tissue or cellular cytosol)
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2. 50/zl of diluted anti-CRBP IgG solution (containing 0.04/zg purified specific IgG and 1.96/zg of preimmune IgG) or diluted anti-CRABP IgG solution (containing 0.02/zg of purified specific IgG and 0.98/xg of preimmune IgG) 3. 300/xl of RIA buffer; incubate for 3-4 hr 4. 50 /A of 125I-labeled CRBP or 125I-labeled CRABP (-20,000 cpm ~25I); incubate 20-24 hr at 4 ° with gentle agitation 5. 25/zl of rabbit antisera against turkey IgG; incubate 12-18 hr at 4 ° with gentle agitation. Samples are centrifuged at 4000 g for 30 min at 4 °, the supernatants are removed by aspiration, and the precipitates are assayed for ~25I. The standard solutions of CRBP or CRABP are prepared from purified CRBP or CRABP diluted in RIA buffer. The concentrations of pure CRBP and CRABP are estimated spectrophotometrically using ~_ll - - E, '~ ~l cm of 14. 26 For both the CRBP RIA and the CRABP RIA, the working ranges of the assays are between 1 and 16 ng of CRBP or CRABP per assay tube. Tissue cytosol preparations are prepared from perfused (with ice-cold 0.9% NaCI) freshly dissected tissues. The dissected tissues are minced, and 500 mg of the tissue mince is added to 3.0 mi RIA buffer and homogenized with a Polytron homogenizer (Brinkmann Instruments) at setting 5 for 30 sec. The tissue homogenates are centrifuged at 105,000 g for 1 hr, and the resulting clear supernatant is diluted appropriately with RIA buffer for assay. Cell pellets containing approximately 10 7 cells are homogenized in 1.0 ml RIA buffer with a Polytron homogenizer on setting 5 for 30 sec. Cytosols from the cell homogenates are prepared by centrifugation of the homogenates at 105,000 g for 1 hr.
Discussion
The procedures described above provide sensitive, specific, and reproducible methods for measuring CRBP and CRABP levels in tissues or isolated cells. Levels of CRBP and CRABP determined with these two RIA procedures, for some tissues and cell types, are given in Table I. For the CRBP RIA, the values for the within-assay and for the between-assay variability are estimated to be 2.7 and 4.8%, respectively. 5 The lower detection limit of the CRBP RIA is 1 ng CRBP/assay tube; hence, it is possible to measure accurately tissue levels as low as 0.07/zg CRBP/g tissue or cellular levels as low as 1 ng CRBP/106 cells. The within-assay and between-assay variabilities for the RIA of CRABP are estimated to be 26 A. C. Ross, Y. I. Takahashi, and D. S. Goodman, J. Biol. Chem. 253, 6591 (1978).
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RECEPTORS, TRANSPORT, AND BINDING PROTEINS
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TABLE I LEVELS OF CRBP AND CRABP IN VARIOUS RAT TISSUES AND CELLS
Tissue or cell
CRBP~
Liverb 40.0 ± 9.7 Small intestineb 10.3 -+ 1.4 Brainb 4.4 ± 0.5 Skinb 3.5 ± 0.6 Fat b 1.2 -+ 0.3 Testis b 17.8 ± 4.8 Seminal vesicle b 2.9 ± 0.7 Hepatic parenchymal cells c 470 ± 238 Hepatic stellate cells c 236 ± 89
CRABP° 1.1 -+ 0.2 0.8 -+ 0.2 1.2 ± 0.3 12.6 ± 2.3 0.2 - 0.1 9.5 -+ 1.1 30.4 ± 7.9 5.6 -+ 3.9 8.7 ± 5.7
Tissue levels of CRBP and CRABP are given as micrograms CRBP or CRABP per gram wet weight of the tissue. Cellular levels of CRBP and CRABP are given as nanograms CRBP or CRABP per 106 cells. All values are given as the mean --+ 1 S.D. Levels of CRBP and CRABP are from Ref. 5. Levels of CRBP and CRABP are from Ref. 20. When these levels are expressed on the basis of cellular protein, the hepatic parenchymal cell levels of CRBP and CRABP are 243 ± 124 ng CRBP/mg protein and 2.9 --- 3.3 ng CRABP/mg protein; hepatic stellate cell levels of CRBP and CRABP are 1256 --- 477 ng CRBP/mg protein and 44 --- 30 ng CRABP/mg protein.
4.1 a n d 5.5%, r e s p e c t i v e l y ) L o w e r d e t e c t i o n limits for the C R A B P R I A are 0 . 0 7 / ~ g C R A B P / g t i s s u e a n d 1 ng C R A B P / 1 0 6 cells. I n c a r r y i n g o u t the a s s a y s for C R B P a n d C R A B P , it is i m p o r t a n t to a d h e r e to the p r o t o c o l s d e s c r i b e d a b o v e . T h e t i m e s a n d t e m p e r a t u r e s o f i n c u b a t i o n s are i m p o r t a n t for the s e n s i t i v i t i e s (affecting the w o r k i n g r a n g e s ) o f e a c h o f the R I A s . T h e p r e p a r a t i o n s o f 125I-labeled C R B P o r 125Il a b e l e d C R A B P s h o u l d h a v e specific a c t i v i t i e s b e t w e e n 20 a n d 40 m C i / m m o l . I f C R B P o r C R A P B are e i t h e r m o r e o r less h e a v i l y l a b e l e d with 1251, the s t a n d a r d d i s p l a c e m e n t c u r v e s are f o u n d to shift to l o w e r s e n s i t i v ity. T h e 125I-labeled C R B P a n d 125I-labeled C R A B P c a n be s t o r e d in 1% B S A at - 2 0 ° for u p to 1 m o n t h . T i s s u e o r cell c y t o s o l p r e p a r a t i o n s c a n b e s t o r e d o v e r n i g h t at 4 ° o r for u p to 14 d a y s at - 8 0 °. S t o r a g e o f c y t o s o l p r e p a r a t i o n s for l o n g e r t h a n 14 d a y s at - 8 0 ° r e s u l t s in a d e c r e a s e in the i m m u n o r e a c t i v e C R B P a n d C R A B P c o n t e n t s o f the c y t o s o l p r e p a r a t i o n s . It is n e c e s s a r y to titer e a c h n e w b a t c h o f s e c o n d a n t i b o d y ( r a b b i t anti-
[28]
X-RAY CRYSTALLOGRAPHICSTUDIESOF RBP
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turkey IgG) employed in the assays, to ensure that this antibody is not present in limiting concentrations. For eight different rabbit anti-turkey IgG antisera examined by this author, 25 p,l of each antisera provided a large excess of anti-turkey IgG antisera; however, this need not always be true. Acknowledgments The author would like to thank Drs. DeWitt S. Goodman and John E. Smith for their help and support. This work was supported by National Institutes of Health Grants DK 05968 and HL 21006.
[28] X - R a y C r y s t a l l o g r a p h i c S t u d i e s on Retinol-Binding Proteins
By MARCIA NEWCOMER and T. ALWYN JONES Introduction Structural information for proteins which interact with retinoids, in their transport or metabolism, or which mediate retinoid function(s) is critical to our understanding of the role(s) of natural or synthetic retinoids in physiology and medicine. X-Ray crystallographic studies of this class of proteins can provide the details we need to understand retinoid recognition at the molecular level. The steps involved in a structural determination can be briefly summarized as follows. (1) The protein must be isolated in relatively large quantity (> 10 mg) with a very high degree of purity. (2) Protein crystals suitable for X-ray diffraction data collection are prepared. (3) X-Ray diffraction data are collected. (4) The "phase problem" is solved by an appropriate method. (5) An electron density map is calculated. (6) A model for the protein structure is built into the electron density. (7) The model is refined. Once a high-resolution structure is determined, the structures of the protein complexed with ligand or ligand analogs are relatively straightforward to determine. The three-dimensional structure of the retinol-binding protein (RBP) from human serum has been determined by the process briefly outlined above. 1 For information with respect to the standard methods in protein structure determination employed, the reader is referred to Volumes 114 i M. E. Newcomer, T. A. Jones, J. ~quist, J. Sundelin, U. Eriksson, L. Rask, and P. A. Peterson, EMBO J. 3, 1451 (1984).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
