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Affinity Labeling of Rat Serum Vitamin D Binding Protein
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 333, No. 1, September 1, pp. 139–144, 1996 Article No. 0374
Affinity Labeling of Rat Serum Vitamin D Binding Protein Narasimha Swamy and Rahul Ray1 Bioorganic and Protein Chemistry, Vitamin D Laboratory, Department of Medicine and Department of Physiology, Boston University School of Medicine, Boston, Massachusetts
Received February 9, 1996, and in revised form June 20, 1996
Vitamin D binding protein (DBP) plays an essential role in the vitamin D hormone endocrine system in sequestering vitamin D3 and its metabolites with high affinity, and transporting them to various target organs and tissues. In the present investigation, 25-hydroxyvitamin D3-3b-(1,2-epoxypropyl)ether (25-OH-D3epoxide) and 25-hydroxyvitamin D3-3b-bromoacetate (25-OH-D3-BE), synthetic analogs of 25-hydroxyvitamin D3 (25-OH-D3), were developed as affinity alkylating reagents for the covalent modification of the 25OH-D3-binding site in rat vitamin D binding protein (rDBP). Competitive radioligand binding assays of 25OH-D3-BE and 25-OH-D3-epoxide with affinity-purified rDBP demonstrated that these analogs displaced 25hydroxy[26(27)-3H]vitamin D3 (3H-25-OH-D3), specifically bound to rDBP, in a dose-dependent fashion. Incubation of rDBP samples with radiolabeled versions of these analogs, i.e., 3H-25-OH-D3-epoxide and 3H-25OH-D3-BE, resulted in the covalent labeling of rDBP. This labeling was largely prevented when incubations were carried out in the presence of an excess of 25OH-D3 , the natural ligand for rDBP. Labeling-specificity by these analogs was further demonstrated by the covalent labeling, inhibited by coincubation with a large excess of 25-OH-D3 , of a single protein band, upon incubating rat serum Cohn IV fraction with 3H-25-OHD3-epoxide and 3H-25-OH-D3-BE. Collectively, these results strongly suggested that 3H-25-OH-D3-epoxide and 3 H-25-OH-D3-BE covalently modified the 25-OH-D3binding site in rDBP. The reagents described in this report could be important in mapping the 25-OH-D3binding pocket in rDBP. q 1996 Academic Press, Inc.
Vitamin D binding protein (DBP)2 or Group Specific Component (Gc) is a genetically polymorphic and 1 To whom correspondence should be addressed at 80 East Concord Street, Boston, MA 02118. Fax: (617) 638-8182. E-mail: Bapi@ bu.edu. 2 Abbreviations used: DBP, vitamin D binding protein; Gc, Group Specific Component; rDBP, rat DBP; hDBP, human DBP; 25-OH-
multifunctional serum glycoprotein (1). DBP binds vitamin D and its metabolites with high specificity and transports them to target tissues (2). In addition, DBP plays a major role in scavenging monomers of actin, in conjunction with plasma gelsolin. DBP thereby prevents actin monomers from polymerizing and clogging arteries during cell injury and lysis (3, 4). DBP binds lipids and fatty acids with high affinity (5, 6). DBP also enhances complement activation on neutrophil chemotaxis by binding to complement C5a and C5a des Arg (7). Moreover, there is evidence to suggest that certain immune responses may be mediated by DBP. For example, DBP has been found to be associated with a variety of cell types including B lymphocytes (8), subpopulations of T lymphocytes (9), and cytotrophoblasts of placental yolk sac (10). In addition, it has been discovered recently that DBP is converted in vivo to a potent macrophage and osteoclast stimulating factor (11, 12). The primary structure of rat DBP (rDBP) has been determined recently. It has been demonstrated that rDBP is highly homologous with the human variety (hDBP) (13). Additionally, DBP, being a member of the albumin gene family, shares a high degree of structural homology with albumin, a-fetoprotein, and afamin (1, 14). All these proteins contain a large number of cysteines (Cys), mostly or all (in the case of DBP) in the disulfide form. Furthermore, these proteins have a coiled structure containing three independent domains. There is strong evidence to suggest that the vitamin D sterol binding and actin binding take place exclusively through N- and C-termini of hDBP, respectively (15–17). The fatty acid binding has also been predicted to take place in domain III of hDBP (18). In the past we have developed photoaffinity labeling reagents for rDBP as a part of our ongoing interest D3 , 25-hydroxyvitamin D3 ; 25-OH-D3-epoxide, 25-hydroxyvitamin D3-3b-(1,2-epoxypropyl)ether; 25-OH-D3-BE, 25-hydroxyvitamin D33b-bromoacetate; DCC, dicyclohexylcarbodiimide; DMAP, 4-N,N*-dimethylaminopyridine. 139
0003-9861/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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in characterizing the binding domain/domains of DBP from different species (19, 20). In the present study we investigated two synthetic analogs of 25-hydroxyvitamin D3 (25-OH-D3), i.e., 25-hydroxyvitamin D3-3b(1,2-epoxypropyl)ether (25-OH-D3-epoxide) and 25-hydroxyvitamin D3-3b-bromoacetate (25-OH-D3-BE) as potential affinity labeling reagents for the 25-OH-D3 binding site in rDBP. MATERIALS AND METHODS 25-Hydroxy[26(27)-3H]vitamin D3 (3H-25-OH-D3) (sp act 21.6 Ci/ mmol) was purchased from Amersham Corp. (Arlington Heights, IL). All other chemicals was purchased from Aldrich Chemical Co. (Milwaukee, WI), except Triton X-100 which was purchased rom Sigma Chemical Co. (St. Louis, MO). 25-OH-D3 was a generous gift from Dr. Richard W. Gray, Amoco Research Corp. (Naperville, IL). 25Hydroxy[26(27)-3H]vitamin D3-3b-(1,2-epoxypropyl)ether (3H-25OH-D3-epoxide) was synthesized according to a method developed by us (21). 25-Hydroxyvitamin D3-3b-[26(27)-3H]bromoacetate (3H-25-OH-D3BE) was synthesized by stirring a solution of 3H-25-OH-D3 (sp act 21.6 Ci/mmol, 10 mCi), vitamin D3 (100 mg), dicyclohexylcarbodiimide (DCC, 3 molar equivalents of vitamin D3), bromoacetic acid (1.2 molar equivalents of vitamin D3), and 4-N,N*-dimethylaminopyridine (DMAP, 0.1 molar equivalent of vitamin D3) in 0.5 ml of anhydrous dichloromethane in an argon atmosphere. After 15 h the reaction mixture was applied to a 1000-mm silica plate (Analtech Co, Vineland, NJ) which was eluted with 33% ethyl acetate in hexane. The radioactive band, corresponding to a standard sample of 25-OH-BE [synthesized by stirring a solution of 25-OH-D3 , bromoacetic acid (1.2 molar equivalents), DCC (3 molar equivalents), and DMAP, 0.1 molar equivalent) in anhydrous dichloromethane, followed by preparative TLC on silica as described above] was isolated. The yield of the product was 3 mCi. HPLC of a sample of 3H-25-OH-D3-BE, mixed with an authentic sample of 25-OH-D3-BE, produced a single radioactive peak corresponding to the uv-active peak of 25-OH-D3-BE (results not shown). Purified rDBP was obtained from pooled rat serum by our recently developed ligand-affinity chromatographic method (22, 23). Briefly, rat serum, diluted (1:1) with TEST buffer (50 mM Tris-HCl, 150 mM sodium chloride, 1.5 mM EDTA, 0.1% Triton X-100 (w/v), pH 8.3), was applied to a 25-OH-D3 –Sepharose affinity column (22). The column was washed thoroughly with the TEST buffer to remove unbound proteins and other materials, followed by the elution of rDBP with aqueous 3 M guanidine-HCl. The pooled guanidine-HCl fractions were dialyzed exhaustively against water, and then against 10 mM phosphate buffer, pH 7.0. The dialysate was applied to a hydroxylapatite column equilibrated with 10 mM phosphate buffer, pH 7. After washing the column with the same buffer, rDBP was eluted with 100 mM phosphate buffer, pH 7. Salt was removed by dialysis against water, followed by lyophilization to produce the pure protein. Purity of this affinity-purified rDBP was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and visualization by staining/destaining with Coomasie brilliant blue. Competitive radioligand-binding assays of 25-OH-D3-epoxide and 25-OH-D3-BE with rDBP. These assays were carried out according to a published procedure (20) with minor modifications. Thus, solutions containing affinity-purified rDBP (400 ng), 3H-25-OH-D3 (0.17 pmol) and 25-OH-D3 (0.99–63.8 pmol) or 25-OH-D3-BE (0.012–5.95 nmol) or 25-OH-D3-epoxide (0.87–56 pmol) in 10 ml of ethanol, and 490 ml of assay buffer (50 mM Tris-HCl, 150 mM sodium chloride, 1.5 mM ethylenediamine tetraacetic acid and 0.1% Triton X-100, pH 8.3) were incubated at 47C for 20 h followed by treatment with ice-cold
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FIG. 1. Denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of affinity-purified rDBP. After the electrophoresis, protein bands were visualized by Coomasie blue staining. Lane 1, Standard molecular weight marker proteins; Lane 2, affinity-purified rDBP.
Dextran-coated charcoal and centrifugation. Supernatants from centrifuged samples were mixed with scintillation cocktail and counted for radioactivity. Affinity labeling of rDBP with 3H-25-OH-D3-epoxide and 3H-25OH-D3-BE. Samples of rDBP (20 mg, 0.4 nmol each) in Tris-HCl buffer, pH 8.3, were incubated with either 3H-25-OH-D3-epoxide (20,000 cpm, 0.93 pmol) or 3H-25-OH-D3-BE (20,000 cpm, 0.93 pmol) alone, or a mixture of 3H-25-OH-D3-epoxide (20,000 cpm) or 3H-25OH-D3-BE (20,000 cpm) and 25-OH-D3(1 mg, 2.5 nmol), dissolved in 5 ml of ethanol. The samples (total volume 50 ml) were incubated initially for 12 h at 47C, then 4 h at 257C. After the incubation, the samples were boiled briefly with electrophoresis sample buffer containing b-mercaptoethanol, and electrophoresed on a 10% SDS gel. A sample containing a mixture of prestained standard molecular weight marker proteins was electrophoresed alongside the experimental samples. After the electrophoresis, the gel was dried and scanned for radioactivity in a Bioscan Imaging Scanner System 200 (Bioscan Inc., Washington, DC). Positions of marker proteins on the dried gel were located visually. Affinity labeling of rat Cohn IV fraction with 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE. Samples of rat Cohn IV fraction (a kind gift from Sigma Chemical Co.), dissolved in Tris buffer, were incubated with either 3H-25-OH-D3-epoxide (20,000 cpm, 0.93 pmol) or 3H-25OH-D3-BE (20,000 cpm, 0.93 pmol) alone, or a mixture of 3H-25-OHD3-epoxide (20,000 cpm) or 3H-25-OH-D3-BE (20,000 cpm) and 25OH-D3(1 mg, 2.5 nmol), dissolved in 5 ml of ethanol. The samples (total volume 50 ml) were incubated initially for 12 h at 47C, then 4 h at 257C. After the incubation, the samples were treated exactly the same way as described earlier, and electrophoressed on a 10% SDS gel. The gel was stained/destained with Coomasie brilliant blue, dried, and subjected to radioactive scanning.
RESULTS AND DISCUSSION
It has been mentioned earlier that rDBP and hDBP share a high degree of amino acid and cDNA sequence
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FIG. 2. Competitive radioligand binding assays of 25-OH-D3 , 25-OH-D3-epoxide, and 25-OH-D3-BE with rDBP. Samples of rDBP were incubated at 47C with 3H-25-OH-D3 (0.17 pmol) and 25-OH-D3 (0.99–63.8 pmol) or 25-OH-D3-BE (0.012–5.95 nmol) or 25-OH-D3-epoxide (0.87–56 pmol) followed by treatment with Dextran-coated charcoal, centrifugation, and radioactive counting.
homologies (1, 13, 24). However, certain functional differences between these proteins have been identified. For example, gonadal steroids have been shown to affect rDBP in a manner opposite to that observed in humans (25). Recently Vieth et al. demonstrated that rat serum DBP has approximately 3.5-times stronger binding affinity for 25-OH-D3 than human serum DBP, and as a result, rat serum exhibited a much lower amount of free 25-OH-D3 than that in human serum (26). It is probable that the above-mentioned difference in 25-OH-D3-binding activities between these proteins may be due to differences in the structural elements of the 25-OH-D3/vitamin D sterol-binding pockets of these proteins. Confirmation of this possibility could potentially be obtained by the identification of the ‘‘contact points’’ within the 25-OH-D3-binding sites in hDBP and rDBP. Recently, our laboratory (15) and that of Haddad et al. (17) have utilized photoaffinity and affinity labeling methods to determine that the vitamin D sterol binding by hDBP is restricted exclusively to the Nterminus of the protein. In addition, we have demonstrated that Trp-145, the only Trp in hDBP, is crucial for 25-OH-D3 binding by hDBP (27). In rDBP, this Trp is replaced by a Phe residue. Hence, this change in a single amino acid may have contributed significantly toward the differential 25-OH-D3-binding activity between these two proteins.
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It is well-documented that, in the photoaffinity labeling process, covalent labeling of an amino acid residue depends on the proximity of that amino acid residue to the highly reactive nitrene or carbene intermediate generated in situ by photolysis (28). In contrast, the affinity labeling process depends entirely on the juxtaposition of a nucleophilic amino acid residue (i.e., Lys, Arg, Cys, Tyr) to the chemically reactive group in the affinity analog of the natural ligand. In essence, the two processes often lead to the ‘‘tagging’’ of different amino acid residues in the ligand-binding pocket. Such information is important for the topographical characterization of the ligand-binding pocket. Earlier we developed two generations of photoaffinity analogs to covalently label rDBP in rat serum (19, 20). In the present study we investigated 25-OH-D3-epoxide and 25-OH-D3-BE, affinity alkylating analogs of 25OH-D3 , to determine their ability to covalently modify the 25-OH-D3-binding site in affinity-purified rDBP. We used our recently developed affinity purification method to purify rDBP from the serum in two steps, as described under Materials and Methods of Ref. (22). The purity of this affinity-purified rDBP sample was determined by SDS–PAGE, which produced a single Coomasie-stained band with an apparent molecular weight of 55 kDa (Fig. 1, Lane 2). Next, we analyzed the binding-affinities of 25-OH-
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FIG. 3. ‘‘Carrier-synthesis’’ of 25-hydroxyvitamin D3-3b-[26(27)3 H]bromoacetate (3H-25-OH-D3-BE).
D3-epoxide and 25-OH-D3-BE to rDBP by competitive radioligand binding assays, in which a fixed amount of 3 H-25-OH-D3 , bound specifically to DBP, was chased with various concentrations of 25-OH-D3 or its analogs, i.e., 25-OH-D3-epoxide or 25-OH-D3-BE. Results of these assays showed that both the analogs displaced 3 H-25-OH-D3 in a dose-dependent manner (Fig. 2). Furthermore, the binding avidity of 25-OH-D3-BE for rDBP was approximately 30 times less than that of 25-OH-D3 , as determined by the half-maximal specific binding of this analog compared to that of 25-OH-D3 . Surprisingly, 25-OH-D3-epoxide was almost as efficient as 25-OH-D3 in displacing 3H-25-OH-D3 from its binding pocket in rDBP, a property similar to that observed with the human counterpart (21). The reason for this discrepancy, which may depend on the molecular size, shape, and polarities of these analogs (compared to those of 25-OH-D3), is unclear at this stage. Synthesis of 14C-25-OH-D3-BE with very low specific activity was reported earlier (17). We, however, concluded that this low specific activity material, with low limit of detection and higher possibility of nonspecific labeling, was unsuitable for our studies, particularly with the ultimate goal of mapping the 25-OH-D3-binding site in rDBP. To circumvent the problem, we designed a method to synthesize 3H-25-OH-D3-BE of high specific activity using commercially available, and ‘‘undiluted’’ 3H-25-OH-D3 (sp act 21.6 Ci/mmol) as the starting material and vitamin D3 as ‘‘carrier’’ (Fig. 3). The desired product (3H-25-OH-D3-BE), obtained in a reasonably high yield, was radiochemically homogeneous as determined by HPLC analysis (results not shown). To determine the capability of 3H-25-OH-D3-epoxide
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and 3H-25-OH-D3-BE to covalently label rDBP, we incubated samples of affinity-purified rDBP with either of these analogs. Results of these experiments, determined by SDS–PAGE/fluorography, demonstrated that both 3H-25-OH-D3-epoxide (Fig. 4, Lane 1) and 3H25-OH-D3-BE (Fig. 5, Lane 1) covalently labeled rDBP. In each case, when the incubation was carried out in the presence of an excess of 25-OH-D3 (2684 molar equivalents), labeling was completely obliterated (Fig. 4, Lane 2, and Fig. 5, Lane 2). These results provided strong evidence that 3H-25-OH-D3-epoxide and 3H-25OH-D3-BE specifically modified the 25-OH-D3-binding site in rDBP. Radioactivity at the bottom of each gel most probably represented the ‘‘free label.’’ We further demonstrated the labeling specificity of 3 H-25-OH-D3-epoxide and 3H-25-OH-D3-BE by incubating samples of rat Cohn IV fraction (Sigma Chemical Co.) with 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE in the presence or in the absence of an excess of 25-
FIG. 4. Affinity labeling of rDBP with 3H-25-OH-D3-epoxide. rDBP samples (20 mg, 0.4 nmol each) were incubated with 3H-25-OH-D3epoxide (20,000 cpm, 0.93 pmol) in the absence (Lane 1) and presence (Lane 2) of an excess of 25-OH-D3 (1 mg, 2.5 nmol). After the incubation, the samples were electrophoresed on a 10% SDS–polyacrylamide gel. After the electrophoresis, the gel was dried and radioactivity was monitored by a radioactivity scanner. Positions of standard molecular weight marker proteins are denoted on the right.
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eral contact points will enable us to develop several recombinant rDBP proteins, point-mutated at those contact points (29). Binding studies of these recombinant proteins with 25-OH-D3 and other vitamin D metabolites would lead to the unequivocal identification of the 25-OH-D3-binding pocket in rDBP. DBP is a multifunctional protein with yet-unknown physiological importance. It has been postulated that ‘‘DBP may be absolutely essential for our survival’’ (1) due to the nonexistence of DBP0 (Gc0) homozygotes despite extensive blood-typing. However, Thornton et al. have recently developed a transgenic mouse model
FIG. 5. Affinity labeling of rDBP with 3H-25-OH-D3-BE. rDBP samples (20 mg, 0.4 nmol each) were incubated with 3H-25-OH-D3-BE (20,000 cpm, 0.93 nmol) in the absence (Lane 1) and presence (Lane 2) of an excess of 25-OH-D3 (1 mg, 2.5 nmol). The incubates were analyzed by SDS–PAGE (10% gel) and radioactivity scanning. Positions of standard molecular weight marker proteins are denoted on the right.
OH-D3 . As shown in Fig. 6B, 3H-25-OH-D3-BE and 3H25-OH-D3-epoxide labeled a single protein band (Fig. 6B, Lanes 1 and 5, respectively) from a mixture of proteins (Fig. 6A, Lanes 1 and 5). Labeling was inhibited completely in both cases by coincubation with an excess of 25-OH-D3 (Fig. 6B, Lanes 2 and 6), further demonstrating the specificity of labeling by 3H-25-OH-D3-BE and 3H-25-OH-D3-epoxide. It is noteworthy that 25-OH-D3-epoxide and 25-OHD3-BE have different reactivities toward amino acids, depending on the pH of the solution, as well as that of the microenvironment of the binding pocket (21). As a result, the two analogs may alkylate different amino acid residues (contact points) within the vitamin D sterol-binding domain of rDBP. Such a situation will potentially allow us to identify several contact points within the 25-OH-D3-binding pocket of rDBP, and ultimately determine the three-dimensional topography of this binding pocket. Additionally, identification of sev-
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FIG. 6. Affinity labeling of rDBP in rat serum Cohn IV fraction with 3H-25-OH-D3-BE and 3H-25-OH-D3-epoxide. Samples of rat serum Cohn IV fraction were incubated with either 3H-25-OH-D3-BE (20,000 cpm) in the absence (Lane 1) or in the presence of 25-OHD3 (1 mg) (Lane 2) or with 3H-25-OH-D3-epoxide (20,000 cpm) in the absence (Lane 5) or in the presence of 25-OH-D3 (1 mg) (Lane 6). Following the incubation, the samples were electrophoresed on a 10% SDS gel. Two samples of affinity-purified rDBP were labeled with 3 H-25-OH-D3-BE or 3H-25-OH-D3-epoxide and electrophoressed in the same gel (Lanes 3 and 7, respectively. Following the electrophoresis, the gel was stained with Coomasie blue (A), destained, and dried by standard procedures, and the positions of radioactive bands in the gel were determined by radioactive scanning (B). Standard molecular weight marker proteins were also electrophoressed in the same gel (Lanes 4 and 8).
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lacking the DBP gene (30). These researchers observed that DBP0 (Gc0) animals were viable and grossly normal. This evidence certainly clouds the argument about the essential nature of DBP. On the other hand, discovery of DBP-macrophage activating factor (DBP-MAF), a posttranslationally modified version of DBP with strong macrophage- and osteoclast-activating properties, has kindled renewed interest in DBP (11). For example, Schneider et al. have recently implicated DBP-MAF in certain inflammatory osteolytic diseases based on results obtained with the in vitro-produced DBP-MAF on osteopetrotic rats (12). Obviously, rat provides an important laboratory model for studying various physiological aspects of DBP and DBP-MAF. However, to use rat as a model, proper understanding of the rat protein is essential. Although the vitamin D sterol-binding property of rDBP has been studied, only scant information about the structure–functional aspect of this binding process and that of others is currently available. The studies described in this report will provide important structure–functional information about the vitamin sterol binding by rDBP. ACKNOWLEDGMENTS The authors thank Dr. Jan K. Blusztaijn for his assistance with fluorography. This work was supported in parts by grants from the National Institutes of Health (DK 44337 and DK 47418).
REFERENCES 1. Cooke, N. E., and Haddad, J. G. (1989) Endocrine Rev. 10, 294– 307. 2. Daiger, D., Schanfield, M. S., and Cavalli-Sforza, L. L. (1975) Proc. Natl. Acad. Sci. USA 72, 2076–2080. 3. Van Baelen, H., Bouillon, R., and DeMoor, P. (1980) J. Biol. Chem. 255, 2270–2272. 4. Haddad, J. G. (1982) Arch. Biochem. Biophys. 213, 538–544. 5. Williams, M. H., Van Alstyne, E. L., and Galbraith, R. M. (1988) Biochem. Biophys. Res. Commun. 153, 1019–1024. 6. Calvo, M., and Ena, J. M. (1989) Biochem. Biophys. Res. Commun. 163, 14–17. 7. Kew, R. R., and Webster, R. O. (1988) J. Clin. Invest. 82, 364– 369.
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8. Petrini, M. D., Emerson, D. L., and Galbraith, R. M. (1983) Nature (London) 306, 73–74. 9. Petrini, M. D., Galbraith, R. M., Emerson, D. L., Nel, A. E., and Arnaud, P. (1985) J. Biol. Chem. 260, 1804–1810. 10. Nester, J. E., McLeod, J. F., Kowalski, M. A., Strauss, I. F. III, and Haddad, J. G. (1987) Endocrinology 120, 1996–2002. 11. Yamamoto, N., and Homma, S. (1991) Proc. Natl. Acad. Sci. USA 88, 8539–8543. 12. Schneider, G. B., Benis, K. A., Flay, N. W., Ireland, R. A., and Popoff, S. N. (1995) Bone 16, 657–662. 13. Cooke, N. E. (1986) J. Biol. Chem. 261, 3441–3450. 14. Lichenstein, H. S., Lyons, D. E., Wurfel, M. M., Johnson, D. A., McGinley, M. D., Leidli, J. C., Trollinger, D. B., Mayer, J. P., Wright, S. D., and Zukowski, M. M. (1994) J. Biol. Chem. 269. 15. Ray, R., Bouillon, R., Van Baelen, H. G., and Holick, M. F. (1991) Biochemistry 30, 7638–7642. 16. Link, R., Kutner, A., Schnoes, H. K., and DeLuca, H. F. (1987) Biochemistry 26, 3957–3964. 17. Haddad, J. G., Hu, Y. Z., Kowalski, M. A., Laramore, C., Ray, K., Robzyk, P., and Cooke, N. E. (1992) Biochemistry 31, 7174– 7181. 18. Ena, J. M., Esteban, C., Perez, M. D., Uriel, J., and Calvo, M. (1989) Biochem. Int. 19, 1–7. 19. Ray, R., Holick, S. A., Hanafin, N., and Holick, M. F. (1986) Biochemistry 25, 4729–4733. 20. Ray, R., Bouillon, R., Van Baelen, H. G., and Holick, M. F. (1991) Biochemistry 30, 4809–4813. 21. Swamy, N., and Ray, R. (1995) Arch. Biochem. Biophys. 319, 504–507. 22. Swamy, N., Roy, R., Chang, R., Brisson, M., and Ray, R. (1995) J. Prot. Exp. Purif. 6, 185–188. 23. Roy, A., and Ray, R. (1995) Steroids 60, 530–533. 24. Ray, K., Wang, X., Zhao, M., and Cooke, N. E. (1991) J. Biol. Chem. 266, 6221–6229. 25. Bouillon, R., Vandoren, G., Van Baelen, H., and De Moor, P. (1978) Endocrinology 102, 1710–1715. 26. Vieth, R., Kessler, M. J., and Pritzker, K. P. H. (1990) Can. J. Physiol. Pharmacol. 68, 1368–1371. 27. Swamy, N., Brisson, M., and Ray, R. (1995) J. Biol. Chem. 270, 2636–2639. 28. Sweet, F. W., and Murdock, G. L. (1987) Endocr. Rev. 8, 154– 184. 29. Swamy, N., and Ray, R. (1995) Abstract presented at the 17th Annual Meeting of the American Society for Bone and Mineral Research, Baltimore, MD. 30. Thornton, P. S., Monks, B., Hu, Y., Haddad, J. G., Liebhaber, S. A., and Cooke, N. E. (1995) J. Bone Min. Res. Supplement 1, August 1995, p. S494. [Abstract T559]
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Vol. 333, No. 1, September 1, pp. 139–144, 1996 Article No. 0374
Affinity Labeling of Rat Serum Vitamin D Binding Protein Narasimha Swamy and Rahul Ray1 Bioorganic and Protein Chemistry, Vitamin D Laboratory, Department of Medicine and Department of Physiology, Boston University School of Medicine, Boston, Massachusetts
Received February 9, 1996, and in revised form June 20, 1996
Vitamin D binding protein (DBP) plays an essential role in the vitamin D hormone endocrine system in sequestering vitamin D3 and its metabolites with high affinity, and transporting them to various target organs and tissues. In the present investigation, 25-hydroxyvitamin D3-3b-(1,2-epoxypropyl)ether (25-OH-D3epoxide) and 25-hydroxyvitamin D3-3b-bromoacetate (25-OH-D3-BE), synthetic analogs of 25-hydroxyvitamin D3 (25-OH-D3), were developed as affinity alkylating reagents for the covalent modification of the 25OH-D3-binding site in rat vitamin D binding protein (rDBP). Competitive radioligand binding assays of 25OH-D3-BE and 25-OH-D3-epoxide with affinity-purified rDBP demonstrated that these analogs displaced 25hydroxy[26(27)-3H]vitamin D3 (3H-25-OH-D3), specifically bound to rDBP, in a dose-dependent fashion. Incubation of rDBP samples with radiolabeled versions of these analogs, i.e., 3H-25-OH-D3-epoxide and 3H-25OH-D3-BE, resulted in the covalent labeling of rDBP. This labeling was largely prevented when incubations were carried out in the presence of an excess of 25OH-D3 , the natural ligand for rDBP. Labeling-specificity by these analogs was further demonstrated by the covalent labeling, inhibited by coincubation with a large excess of 25-OH-D3 , of a single protein band, upon incubating rat serum Cohn IV fraction with 3H-25-OHD3-epoxide and 3H-25-OH-D3-BE. Collectively, these results strongly suggested that 3H-25-OH-D3-epoxide and 3 H-25-OH-D3-BE covalently modified the 25-OH-D3binding site in rDBP. The reagents described in this report could be important in mapping the 25-OH-D3binding pocket in rDBP. q 1996 Academic Press, Inc.
Vitamin D binding protein (DBP)2 or Group Specific Component (Gc) is a genetically polymorphic and 1 To whom correspondence should be addressed at 80 East Concord Street, Boston, MA 02118. Fax: (617) 638-8182. E-mail: Bapi@ bu.edu. 2 Abbreviations used: DBP, vitamin D binding protein; Gc, Group Specific Component; rDBP, rat DBP; hDBP, human DBP; 25-OH-
multifunctional serum glycoprotein (1). DBP binds vitamin D and its metabolites with high specificity and transports them to target tissues (2). In addition, DBP plays a major role in scavenging monomers of actin, in conjunction with plasma gelsolin. DBP thereby prevents actin monomers from polymerizing and clogging arteries during cell injury and lysis (3, 4). DBP binds lipids and fatty acids with high affinity (5, 6). DBP also enhances complement activation on neutrophil chemotaxis by binding to complement C5a and C5a des Arg (7). Moreover, there is evidence to suggest that certain immune responses may be mediated by DBP. For example, DBP has been found to be associated with a variety of cell types including B lymphocytes (8), subpopulations of T lymphocytes (9), and cytotrophoblasts of placental yolk sac (10). In addition, it has been discovered recently that DBP is converted in vivo to a potent macrophage and osteoclast stimulating factor (11, 12). The primary structure of rat DBP (rDBP) has been determined recently. It has been demonstrated that rDBP is highly homologous with the human variety (hDBP) (13). Additionally, DBP, being a member of the albumin gene family, shares a high degree of structural homology with albumin, a-fetoprotein, and afamin (1, 14). All these proteins contain a large number of cysteines (Cys), mostly or all (in the case of DBP) in the disulfide form. Furthermore, these proteins have a coiled structure containing three independent domains. There is strong evidence to suggest that the vitamin D sterol binding and actin binding take place exclusively through N- and C-termini of hDBP, respectively (15–17). The fatty acid binding has also been predicted to take place in domain III of hDBP (18). In the past we have developed photoaffinity labeling reagents for rDBP as a part of our ongoing interest D3 , 25-hydroxyvitamin D3 ; 25-OH-D3-epoxide, 25-hydroxyvitamin D3-3b-(1,2-epoxypropyl)ether; 25-OH-D3-BE, 25-hydroxyvitamin D33b-bromoacetate; DCC, dicyclohexylcarbodiimide; DMAP, 4-N,N*-dimethylaminopyridine. 139
0003-9861/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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in characterizing the binding domain/domains of DBP from different species (19, 20). In the present study we investigated two synthetic analogs of 25-hydroxyvitamin D3 (25-OH-D3), i.e., 25-hydroxyvitamin D3-3b(1,2-epoxypropyl)ether (25-OH-D3-epoxide) and 25-hydroxyvitamin D3-3b-bromoacetate (25-OH-D3-BE) as potential affinity labeling reagents for the 25-OH-D3 binding site in rDBP. MATERIALS AND METHODS 25-Hydroxy[26(27)-3H]vitamin D3 (3H-25-OH-D3) (sp act 21.6 Ci/ mmol) was purchased from Amersham Corp. (Arlington Heights, IL). All other chemicals was purchased from Aldrich Chemical Co. (Milwaukee, WI), except Triton X-100 which was purchased rom Sigma Chemical Co. (St. Louis, MO). 25-OH-D3 was a generous gift from Dr. Richard W. Gray, Amoco Research Corp. (Naperville, IL). 25Hydroxy[26(27)-3H]vitamin D3-3b-(1,2-epoxypropyl)ether (3H-25OH-D3-epoxide) was synthesized according to a method developed by us (21). 25-Hydroxyvitamin D3-3b-[26(27)-3H]bromoacetate (3H-25-OH-D3BE) was synthesized by stirring a solution of 3H-25-OH-D3 (sp act 21.6 Ci/mmol, 10 mCi), vitamin D3 (100 mg), dicyclohexylcarbodiimide (DCC, 3 molar equivalents of vitamin D3), bromoacetic acid (1.2 molar equivalents of vitamin D3), and 4-N,N*-dimethylaminopyridine (DMAP, 0.1 molar equivalent of vitamin D3) in 0.5 ml of anhydrous dichloromethane in an argon atmosphere. After 15 h the reaction mixture was applied to a 1000-mm silica plate (Analtech Co, Vineland, NJ) which was eluted with 33% ethyl acetate in hexane. The radioactive band, corresponding to a standard sample of 25-OH-BE [synthesized by stirring a solution of 25-OH-D3 , bromoacetic acid (1.2 molar equivalents), DCC (3 molar equivalents), and DMAP, 0.1 molar equivalent) in anhydrous dichloromethane, followed by preparative TLC on silica as described above] was isolated. The yield of the product was 3 mCi. HPLC of a sample of 3H-25-OH-D3-BE, mixed with an authentic sample of 25-OH-D3-BE, produced a single radioactive peak corresponding to the uv-active peak of 25-OH-D3-BE (results not shown). Purified rDBP was obtained from pooled rat serum by our recently developed ligand-affinity chromatographic method (22, 23). Briefly, rat serum, diluted (1:1) with TEST buffer (50 mM Tris-HCl, 150 mM sodium chloride, 1.5 mM EDTA, 0.1% Triton X-100 (w/v), pH 8.3), was applied to a 25-OH-D3 –Sepharose affinity column (22). The column was washed thoroughly with the TEST buffer to remove unbound proteins and other materials, followed by the elution of rDBP with aqueous 3 M guanidine-HCl. The pooled guanidine-HCl fractions were dialyzed exhaustively against water, and then against 10 mM phosphate buffer, pH 7.0. The dialysate was applied to a hydroxylapatite column equilibrated with 10 mM phosphate buffer, pH 7. After washing the column with the same buffer, rDBP was eluted with 100 mM phosphate buffer, pH 7. Salt was removed by dialysis against water, followed by lyophilization to produce the pure protein. Purity of this affinity-purified rDBP was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and visualization by staining/destaining with Coomasie brilliant blue. Competitive radioligand-binding assays of 25-OH-D3-epoxide and 25-OH-D3-BE with rDBP. These assays were carried out according to a published procedure (20) with minor modifications. Thus, solutions containing affinity-purified rDBP (400 ng), 3H-25-OH-D3 (0.17 pmol) and 25-OH-D3 (0.99–63.8 pmol) or 25-OH-D3-BE (0.012–5.95 nmol) or 25-OH-D3-epoxide (0.87–56 pmol) in 10 ml of ethanol, and 490 ml of assay buffer (50 mM Tris-HCl, 150 mM sodium chloride, 1.5 mM ethylenediamine tetraacetic acid and 0.1% Triton X-100, pH 8.3) were incubated at 47C for 20 h followed by treatment with ice-cold
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FIG. 1. Denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of affinity-purified rDBP. After the electrophoresis, protein bands were visualized by Coomasie blue staining. Lane 1, Standard molecular weight marker proteins; Lane 2, affinity-purified rDBP.
Dextran-coated charcoal and centrifugation. Supernatants from centrifuged samples were mixed with scintillation cocktail and counted for radioactivity. Affinity labeling of rDBP with 3H-25-OH-D3-epoxide and 3H-25OH-D3-BE. Samples of rDBP (20 mg, 0.4 nmol each) in Tris-HCl buffer, pH 8.3, were incubated with either 3H-25-OH-D3-epoxide (20,000 cpm, 0.93 pmol) or 3H-25-OH-D3-BE (20,000 cpm, 0.93 pmol) alone, or a mixture of 3H-25-OH-D3-epoxide (20,000 cpm) or 3H-25OH-D3-BE (20,000 cpm) and 25-OH-D3(1 mg, 2.5 nmol), dissolved in 5 ml of ethanol. The samples (total volume 50 ml) were incubated initially for 12 h at 47C, then 4 h at 257C. After the incubation, the samples were boiled briefly with electrophoresis sample buffer containing b-mercaptoethanol, and electrophoresed on a 10% SDS gel. A sample containing a mixture of prestained standard molecular weight marker proteins was electrophoresed alongside the experimental samples. After the electrophoresis, the gel was dried and scanned for radioactivity in a Bioscan Imaging Scanner System 200 (Bioscan Inc., Washington, DC). Positions of marker proteins on the dried gel were located visually. Affinity labeling of rat Cohn IV fraction with 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE. Samples of rat Cohn IV fraction (a kind gift from Sigma Chemical Co.), dissolved in Tris buffer, were incubated with either 3H-25-OH-D3-epoxide (20,000 cpm, 0.93 pmol) or 3H-25OH-D3-BE (20,000 cpm, 0.93 pmol) alone, or a mixture of 3H-25-OHD3-epoxide (20,000 cpm) or 3H-25-OH-D3-BE (20,000 cpm) and 25OH-D3(1 mg, 2.5 nmol), dissolved in 5 ml of ethanol. The samples (total volume 50 ml) were incubated initially for 12 h at 47C, then 4 h at 257C. After the incubation, the samples were treated exactly the same way as described earlier, and electrophoressed on a 10% SDS gel. The gel was stained/destained with Coomasie brilliant blue, dried, and subjected to radioactive scanning.
RESULTS AND DISCUSSION
It has been mentioned earlier that rDBP and hDBP share a high degree of amino acid and cDNA sequence
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FIG. 2. Competitive radioligand binding assays of 25-OH-D3 , 25-OH-D3-epoxide, and 25-OH-D3-BE with rDBP. Samples of rDBP were incubated at 47C with 3H-25-OH-D3 (0.17 pmol) and 25-OH-D3 (0.99–63.8 pmol) or 25-OH-D3-BE (0.012–5.95 nmol) or 25-OH-D3-epoxide (0.87–56 pmol) followed by treatment with Dextran-coated charcoal, centrifugation, and radioactive counting.
homologies (1, 13, 24). However, certain functional differences between these proteins have been identified. For example, gonadal steroids have been shown to affect rDBP in a manner opposite to that observed in humans (25). Recently Vieth et al. demonstrated that rat serum DBP has approximately 3.5-times stronger binding affinity for 25-OH-D3 than human serum DBP, and as a result, rat serum exhibited a much lower amount of free 25-OH-D3 than that in human serum (26). It is probable that the above-mentioned difference in 25-OH-D3-binding activities between these proteins may be due to differences in the structural elements of the 25-OH-D3/vitamin D sterol-binding pockets of these proteins. Confirmation of this possibility could potentially be obtained by the identification of the ‘‘contact points’’ within the 25-OH-D3-binding sites in hDBP and rDBP. Recently, our laboratory (15) and that of Haddad et al. (17) have utilized photoaffinity and affinity labeling methods to determine that the vitamin D sterol binding by hDBP is restricted exclusively to the Nterminus of the protein. In addition, we have demonstrated that Trp-145, the only Trp in hDBP, is crucial for 25-OH-D3 binding by hDBP (27). In rDBP, this Trp is replaced by a Phe residue. Hence, this change in a single amino acid may have contributed significantly toward the differential 25-OH-D3-binding activity between these two proteins.
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It is well-documented that, in the photoaffinity labeling process, covalent labeling of an amino acid residue depends on the proximity of that amino acid residue to the highly reactive nitrene or carbene intermediate generated in situ by photolysis (28). In contrast, the affinity labeling process depends entirely on the juxtaposition of a nucleophilic amino acid residue (i.e., Lys, Arg, Cys, Tyr) to the chemically reactive group in the affinity analog of the natural ligand. In essence, the two processes often lead to the ‘‘tagging’’ of different amino acid residues in the ligand-binding pocket. Such information is important for the topographical characterization of the ligand-binding pocket. Earlier we developed two generations of photoaffinity analogs to covalently label rDBP in rat serum (19, 20). In the present study we investigated 25-OH-D3-epoxide and 25-OH-D3-BE, affinity alkylating analogs of 25OH-D3 , to determine their ability to covalently modify the 25-OH-D3-binding site in affinity-purified rDBP. We used our recently developed affinity purification method to purify rDBP from the serum in two steps, as described under Materials and Methods of Ref. (22). The purity of this affinity-purified rDBP sample was determined by SDS–PAGE, which produced a single Coomasie-stained band with an apparent molecular weight of 55 kDa (Fig. 1, Lane 2). Next, we analyzed the binding-affinities of 25-OH-
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FIG. 3. ‘‘Carrier-synthesis’’ of 25-hydroxyvitamin D3-3b-[26(27)3 H]bromoacetate (3H-25-OH-D3-BE).
D3-epoxide and 25-OH-D3-BE to rDBP by competitive radioligand binding assays, in which a fixed amount of 3 H-25-OH-D3 , bound specifically to DBP, was chased with various concentrations of 25-OH-D3 or its analogs, i.e., 25-OH-D3-epoxide or 25-OH-D3-BE. Results of these assays showed that both the analogs displaced 3 H-25-OH-D3 in a dose-dependent manner (Fig. 2). Furthermore, the binding avidity of 25-OH-D3-BE for rDBP was approximately 30 times less than that of 25-OH-D3 , as determined by the half-maximal specific binding of this analog compared to that of 25-OH-D3 . Surprisingly, 25-OH-D3-epoxide was almost as efficient as 25-OH-D3 in displacing 3H-25-OH-D3 from its binding pocket in rDBP, a property similar to that observed with the human counterpart (21). The reason for this discrepancy, which may depend on the molecular size, shape, and polarities of these analogs (compared to those of 25-OH-D3), is unclear at this stage. Synthesis of 14C-25-OH-D3-BE with very low specific activity was reported earlier (17). We, however, concluded that this low specific activity material, with low limit of detection and higher possibility of nonspecific labeling, was unsuitable for our studies, particularly with the ultimate goal of mapping the 25-OH-D3-binding site in rDBP. To circumvent the problem, we designed a method to synthesize 3H-25-OH-D3-BE of high specific activity using commercially available, and ‘‘undiluted’’ 3H-25-OH-D3 (sp act 21.6 Ci/mmol) as the starting material and vitamin D3 as ‘‘carrier’’ (Fig. 3). The desired product (3H-25-OH-D3-BE), obtained in a reasonably high yield, was radiochemically homogeneous as determined by HPLC analysis (results not shown). To determine the capability of 3H-25-OH-D3-epoxide
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and 3H-25-OH-D3-BE to covalently label rDBP, we incubated samples of affinity-purified rDBP with either of these analogs. Results of these experiments, determined by SDS–PAGE/fluorography, demonstrated that both 3H-25-OH-D3-epoxide (Fig. 4, Lane 1) and 3H25-OH-D3-BE (Fig. 5, Lane 1) covalently labeled rDBP. In each case, when the incubation was carried out in the presence of an excess of 25-OH-D3 (2684 molar equivalents), labeling was completely obliterated (Fig. 4, Lane 2, and Fig. 5, Lane 2). These results provided strong evidence that 3H-25-OH-D3-epoxide and 3H-25OH-D3-BE specifically modified the 25-OH-D3-binding site in rDBP. Radioactivity at the bottom of each gel most probably represented the ‘‘free label.’’ We further demonstrated the labeling specificity of 3 H-25-OH-D3-epoxide and 3H-25-OH-D3-BE by incubating samples of rat Cohn IV fraction (Sigma Chemical Co.) with 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE in the presence or in the absence of an excess of 25-
FIG. 4. Affinity labeling of rDBP with 3H-25-OH-D3-epoxide. rDBP samples (20 mg, 0.4 nmol each) were incubated with 3H-25-OH-D3epoxide (20,000 cpm, 0.93 pmol) in the absence (Lane 1) and presence (Lane 2) of an excess of 25-OH-D3 (1 mg, 2.5 nmol). After the incubation, the samples were electrophoresed on a 10% SDS–polyacrylamide gel. After the electrophoresis, the gel was dried and radioactivity was monitored by a radioactivity scanner. Positions of standard molecular weight marker proteins are denoted on the right.
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eral contact points will enable us to develop several recombinant rDBP proteins, point-mutated at those contact points (29). Binding studies of these recombinant proteins with 25-OH-D3 and other vitamin D metabolites would lead to the unequivocal identification of the 25-OH-D3-binding pocket in rDBP. DBP is a multifunctional protein with yet-unknown physiological importance. It has been postulated that ‘‘DBP may be absolutely essential for our survival’’ (1) due to the nonexistence of DBP0 (Gc0) homozygotes despite extensive blood-typing. However, Thornton et al. have recently developed a transgenic mouse model
FIG. 5. Affinity labeling of rDBP with 3H-25-OH-D3-BE. rDBP samples (20 mg, 0.4 nmol each) were incubated with 3H-25-OH-D3-BE (20,000 cpm, 0.93 nmol) in the absence (Lane 1) and presence (Lane 2) of an excess of 25-OH-D3 (1 mg, 2.5 nmol). The incubates were analyzed by SDS–PAGE (10% gel) and radioactivity scanning. Positions of standard molecular weight marker proteins are denoted on the right.
OH-D3 . As shown in Fig. 6B, 3H-25-OH-D3-BE and 3H25-OH-D3-epoxide labeled a single protein band (Fig. 6B, Lanes 1 and 5, respectively) from a mixture of proteins (Fig. 6A, Lanes 1 and 5). Labeling was inhibited completely in both cases by coincubation with an excess of 25-OH-D3 (Fig. 6B, Lanes 2 and 6), further demonstrating the specificity of labeling by 3H-25-OH-D3-BE and 3H-25-OH-D3-epoxide. It is noteworthy that 25-OH-D3-epoxide and 25-OHD3-BE have different reactivities toward amino acids, depending on the pH of the solution, as well as that of the microenvironment of the binding pocket (21). As a result, the two analogs may alkylate different amino acid residues (contact points) within the vitamin D sterol-binding domain of rDBP. Such a situation will potentially allow us to identify several contact points within the 25-OH-D3-binding pocket of rDBP, and ultimately determine the three-dimensional topography of this binding pocket. Additionally, identification of sev-
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FIG. 6. Affinity labeling of rDBP in rat serum Cohn IV fraction with 3H-25-OH-D3-BE and 3H-25-OH-D3-epoxide. Samples of rat serum Cohn IV fraction were incubated with either 3H-25-OH-D3-BE (20,000 cpm) in the absence (Lane 1) or in the presence of 25-OHD3 (1 mg) (Lane 2) or with 3H-25-OH-D3-epoxide (20,000 cpm) in the absence (Lane 5) or in the presence of 25-OH-D3 (1 mg) (Lane 6). Following the incubation, the samples were electrophoresed on a 10% SDS gel. Two samples of affinity-purified rDBP were labeled with 3 H-25-OH-D3-BE or 3H-25-OH-D3-epoxide and electrophoressed in the same gel (Lanes 3 and 7, respectively. Following the electrophoresis, the gel was stained with Coomasie blue (A), destained, and dried by standard procedures, and the positions of radioactive bands in the gel were determined by radioactive scanning (B). Standard molecular weight marker proteins were also electrophoressed in the same gel (Lanes 4 and 8).
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lacking the DBP gene (30). These researchers observed that DBP0 (Gc0) animals were viable and grossly normal. This evidence certainly clouds the argument about the essential nature of DBP. On the other hand, discovery of DBP-macrophage activating factor (DBP-MAF), a posttranslationally modified version of DBP with strong macrophage- and osteoclast-activating properties, has kindled renewed interest in DBP (11). For example, Schneider et al. have recently implicated DBP-MAF in certain inflammatory osteolytic diseases based on results obtained with the in vitro-produced DBP-MAF on osteopetrotic rats (12). Obviously, rat provides an important laboratory model for studying various physiological aspects of DBP and DBP-MAF. However, to use rat as a model, proper understanding of the rat protein is essential. Although the vitamin D sterol-binding property of rDBP has been studied, only scant information about the structure–functional aspect of this binding process and that of others is currently available. The studies described in this report will provide important structure–functional information about the vitamin sterol binding by rDBP. ACKNOWLEDGMENTS The authors thank Dr. Jan K. Blusztaijn for his assistance with fluorography. This work was supported in parts by grants from the National Institutes of Health (DK 44337 and DK 47418).
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8. Petrini, M. D., Emerson, D. L., and Galbraith, R. M. (1983) Nature (London) 306, 73–74. 9. Petrini, M. D., Galbraith, R. M., Emerson, D. L., Nel, A. E., and Arnaud, P. (1985) J. Biol. Chem. 260, 1804–1810. 10. Nester, J. E., McLeod, J. F., Kowalski, M. A., Strauss, I. F. III, and Haddad, J. G. (1987) Endocrinology 120, 1996–2002. 11. Yamamoto, N., and Homma, S. (1991) Proc. Natl. Acad. Sci. USA 88, 8539–8543. 12. Schneider, G. B., Benis, K. A., Flay, N. W., Ireland, R. A., and Popoff, S. N. (1995) Bone 16, 657–662. 13. Cooke, N. E. (1986) J. Biol. Chem. 261, 3441–3450. 14. Lichenstein, H. S., Lyons, D. E., Wurfel, M. M., Johnson, D. A., McGinley, M. D., Leidli, J. C., Trollinger, D. B., Mayer, J. P., Wright, S. D., and Zukowski, M. M. (1994) J. Biol. Chem. 269. 15. Ray, R., Bouillon, R., Van Baelen, H. G., and Holick, M. F. (1991) Biochemistry 30, 7638–7642. 16. Link, R., Kutner, A., Schnoes, H. K., and DeLuca, H. F. (1987) Biochemistry 26, 3957–3964. 17. Haddad, J. G., Hu, Y. Z., Kowalski, M. A., Laramore, C., Ray, K., Robzyk, P., and Cooke, N. E. (1992) Biochemistry 31, 7174– 7181. 18. Ena, J. M., Esteban, C., Perez, M. D., Uriel, J., and Calvo, M. (1989) Biochem. Int. 19, 1–7. 19. Ray, R., Holick, S. A., Hanafin, N., and Holick, M. F. (1986) Biochemistry 25, 4729–4733. 20. Ray, R., Bouillon, R., Van Baelen, H. G., and Holick, M. F. (1991) Biochemistry 30, 4809–4813. 21. Swamy, N., and Ray, R. (1995) Arch. Biochem. Biophys. 319, 504–507. 22. Swamy, N., Roy, R., Chang, R., Brisson, M., and Ray, R. (1995) J. Prot. Exp. Purif. 6, 185–188. 23. Roy, A., and Ray, R. (1995) Steroids 60, 530–533. 24. Ray, K., Wang, X., Zhao, M., and Cooke, N. E. (1991) J. Biol. Chem. 266, 6221–6229. 25. Bouillon, R., Vandoren, G., Van Baelen, H., and De Moor, P. (1978) Endocrinology 102, 1710–1715. 26. Vieth, R., Kessler, M. J., and Pritzker, K. P. H. (1990) Can. J. Physiol. Pharmacol. 68, 1368–1371. 27. Swamy, N., Brisson, M., and Ray, R. (1995) J. Biol. Chem. 270, 2636–2639. 28. Sweet, F. W., and Murdock, G. L. (1987) Endocr. Rev. 8, 154– 184. 29. Swamy, N., and Ray, R. (1995) Abstract presented at the 17th Annual Meeting of the American Society for Bone and Mineral Research, Baltimore, MD. 30. Thornton, P. S., Monks, B., Hu, Y., Haddad, J. G., Liebhaber, S. A., and Cooke, N. E. (1995) J. Bone Min. Res. Supplement 1, August 1995, p. S494. [Abstract T559]
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