- Email: [email protected]
Application of Photoaffinity Labeling with [11,12-3H]All-trans-Retinoic Acid to Characterization of Rat Liver Microsomal UDP-Glucuronosyltransferase(s) with Activity toward Retinoic Acid
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
230, 497–500 (1997)
RC965992
Application of Photoaffinity Labeling with [11,12-3H]Alltrans-Retinoic Acid to Characterization of Rat Liver Microsomal UDP-Glucuronosyltransferase(s) with Activity toward Retinoic Acid Joanna M. Little1 and Anna Radominska Division of Gastroenterology, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Received December 9, 1996
[3H]All-trans-retinoic acid has been shown to be an effective photoaffinity label for microsomal UDP-glucuronosyltransferases. Labeling of rat liver microsomal proteins with [3H]all-trans-retinoic acid and [32P]5-azido-UDP-glucuronic acid has shown that at least one protein in the 50-56 kDa mass range encompassing the UDP-glucuronosyltransferases photoincorporated both probes. The fraction of solubilized microsomal protein eluted from a UDP-hexanolamine affinity column with 50 mM UDP-glucuronic acid contained two protein bands, both of which photoincorporated [3H]alltrans-retinoic acid and were detected on Western blot by anti-UDP-glucuronosyltransferase antibodies. Enzymatic glucuronidation activity toward atRA in the same fraction was enriched five-fold over that of native or solubilized microsomes. q 1997 Academic Press
Glucuronidation is generally considered to be a reaction of detoxification and elimination. UDP-glucuronosyltransferases (UGTs, EC 2.4.1.17), the enzymes responsible for the glucuronidation reaction, have also been found to play a powerful role in the metabolism of endogenous substances as well as the detoxification of drugs and other xenobiotics. Retinoic acid, a physiologically and pharmacologically important compound, can be metabolized and excreted in the form of an acyl glucuronide. Retinoyl-b-glucuronide was first identi1 To whom correspondence should be addressed at Division of Gastroenterology, University of Arkansas for Medical Science, 4301 W. Markham, Slot 567, Little Rock, AR 72205. Fax: 501-686-6248. Abbreviations: UGT: UDP-glucuronosyltransferase; atRA: all trans-retinoic acid; atRAG: all trans retinoyl-b-glucuronide; [32P]5N3UDP-GlcUA: [b-32P]5-azido-UDP-glucuronic acid; UDP-GlcUA: UDP-glucuronic acid; SDS-PAGE: SDS-polyacrylamide gel electrophoresis; DTT: dithiothreitol; PC: phosphatidyl choline; DTT: dithiothreitol.
fied thirty years ago as a naturally occurring product of the in vivo metabolism of atRA in rats (1). Since that time, in vivo glucuronidation of atRA administered to rats has been confirmed (2-4), in vitro glucuronidation of atRA has been demonstrated in rat liver microsomes (5-7) and rat kidney and intestinal microsomes as well (5) and [11-3H]atRAG has been chemically synthesized and its in vivo metabolism in the rat has been studied (8). In addition, atRAG has been shown to be an endogenous component of human blood (9). This study was prompted by the fact that scant attention has been paid to the characterization or identification of the UGT(s) responsible for the conjugation reaction. We have applied [32P]5N3UDP-GlcUA photoaffinity labeling, which has proven to be very effective in characterizing rat and human liver microsomal UGTs as a group (10-14) and the recently published method of Bernstein et al (15) for direct photoaffinity labeling with [11,12-3H]atRA to the identification of the UGT(s) in rat liver microsomes responsible for RA glucuronidation. Experiments have shown that at least one rat liver microsomal protein in the 50-56 kDa molecular mass range known to contain the UGTs can be photolabeled with both [3H]atRA, a probe for the aglycone binding site, and [32P]5N3UDP-GlcUA, a UDP-GlcUA binding site directed photoaffinity analog. Additionally, two protein bands in the 50-56 kDa range recovered in fractions eluted from a UDP-affinity column with UDPGlcUA were found to photoincorporate [3H]atRA and to be detected by an anti-UGT antibody on Western blots. Enzymatic glucuronidation of RA by these same fractions was found to be 5-fold enriched over that of the solubilized microsomal proteins. MATERIALS AND METHODS Materials. [11,12-3H]atRA was purchased from Dupont-New England Nuclear (Boston, MA). [32P]5N3-UDP-GlcUA was synthesized
497
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 5992
/
6917$$$$21
12-28-96 07:28:12
bbrcg
AP: BBRC
Vol. 230, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and purified as previously described (10, 16). [32P]Pi was from ICN (Costa Mesa, CA). Brij 58, UDP-GlcUA, unlabeled atRA and saccharolactone were purchased from Sigma Chemical Co. (St. Louis, MO). UDP-hexanolamine (Sigma) was linked to cyanogen bromide activated Sepharose 4B (Sigma) as previously described (17). Microsome preparation and solubilization. Male Sprague-Dawley rats (200-250 g) were used and microsomes prepared as described in (18), except that they were not further subfractionated into rough and smooth ER fractions. For solubilization of membrane proteins, microsomes as prepared above were suspended in 10 mM Tris-HCl, pH 7.5 containing 0.5 M KCl, 0.05 mg/ml PC (from egg yolk, Sigma) and 10% glycerol (w/v) at a protein concentration of 3-5 mg/ml. CHAPS was added to a final concentration of 4 mM and the suspension was incubated on ice, with stirring, for one hour followed by centrifugation at 105,000 1 g for one hour. The supernatant was removed and stored at 0207C until use. All protein determinations were carried out using the BioRad protein assay kit (BioRad, La Jolla, CA) with bovine serum albumin as standard. Photoaffinity labeling. The standard protocol for photolabeling of rat liver microsomes with [32P]5N3UDP-GlcUA has been described in detail (11-13, 19). Photolabeling with [11,12-3H]RA was done using the method of Bernstein et al (15) modified as follows. For studies with detergent activated microsomes, [3H]RA (solubilized in micellar form with Triton-X100) was added to rat liver microsomal protein (100 mg) in 100 mM HEPES, pH 7.0 containing 5 mM MgCl2 to a final concentration of 2 mM (2.0 mCi) [3H]RA and 0.05% Triton in a total volume of 25 ml. The reaction was incubated on ice for 10 min followed by irradiation with a hand-held long-wave UV lamp (366 nm, UVP-21, Ultraviolet Products, Inc., San Gabriel, CA) for 15 min on ice. All experiments involving RA were carried out under yellow light. All photolabeling experiments were stopped with 10% TCA (150 ml) and processed for SDS-PAGE on 10% gels (20) as previously described (19). Gels were stained, dried and subjected to autoradiography for 1-7 days. Gels with proteins labeled with [3H]RA were treated with Autofluor (National Diagnostics, Manville, NJ) according to the manufacturer’s directions prior to drying. Autoradiographs were analyzed by densitometry with a BioRad Imaging Densitometer (BioRad, La Jolla, CA). In some cases, proteins were electroblotted from gels to nitrocellulose and subjected to Western blot analysis using the method of Towbin et al (21). Blotted proteins were probed with a rat anti-androsterone UGT antibody, a generous gift from M. Green and Dr. T. Tephly, University of Iowa, Iowa City, IA. UDP-hexanolamine-Sepharose affinity chromatography. All of the following manipulations were carried out at 47C. UDP-hexanolamine-Sepharose was equilibrated with 20 mM HEPES, pH 6.5 containing 10% glycerol, 0.05% CHAPS, 0.1 mM DTT and 25 mM KCl. Solubilized rat liver microsomal protein (approximately 2 mg, prepared as described above) was diluted with 5 ml of equilibration buffer supplemented with 5 mM MgCl2 and 0.05 mg/ml PC (Buffer B). This sample was mixed with 1 ml of UDP-hexanolamine-Sepharose in a small column (BioRad Poly-Prep Chromatography Column), both ends of the column were sealed, and sample and resin were gently shaken overnight. After the resin had been allowed to settle in the column, the sample was allowed to flow through the column under gravity at a flow rate not exceeding 0.5 ml/min. The effluent was collected as a single fraction (flow-through). The column was washed with 50 ml Buffer B, collecting 5 ml fractions, and protein bound to the column was eluted with 5 ml of 50 mM in Buffer B, collected as a single fraction. Fractions were concentrated using Centricon 30 ultrafilters (Amicon, Beverly, MA). Duplicate aliquots of microsomal proteins, solubilized microsomes and concentrated fractions were photolabeled with [3H]atRA as described above and subjected to SDS-PAGE. One gel was processed for autoradiography and the other for Western blotting as described above. Additional aliquots were assayed for enzymatic glucuronidation activity towards RA as described below.
FIG. 1. Photoaffinity labeling of rat liver microsomes with 40 mM [32P]5N3UDP-GlcUA or 2 mM [3H]atRA. Lanes 1-3: photolabeling with [32P]5N3UDP-GlcUA; lane 1: no UV irradiation; lane 2: UV irradiation; lane 3: UV irradiation, with 0.4 mM UDP-GlcUA. Lanes 46: photolabeling with [3H]atRA; lane 4: no UV irradiation; lane 5: UV irradiation; lane 6: UV irradiation, with 20 mM atRA.
Enzymatic assays. Enzymatic retinoid glucuronidation activity in rat liver microsomes was determined as previously described (22). The detergent Brij-58 (final concentration, 0.05%) was used both to solubilize the hydrophobic substrate in micellar form and to activate the enzymatic activity of the microsomal UGTs. All manipulations were carried out under yellow light.
RESULTS Photoaffinity labeling of rat liver microsomes with [32P]5N3UDP-GlcUA and [3H]atRA. The autoradiograph in Figure 1 shows the results of a representative experiment in which rat liver microsomes were photolabeled with either [32P]5N3UDP-GlcUA (lanes 1-3) or [3H]atRA (lanes 4-6) followed by SDS-PAGE separation of microsomal proteins. At least two protein bands (approximately 52 and 54 kDa) in the 50-56 kDa range known to encompass the UGTs (10) were photolabeled with [32P]5N3UDP-GlcUA only after UV irradiation (compare Fig. 1, lanes 1 and 2, without and with UV irradiation, respectively) and the photoincorporation was effectively inhibited by unlabeled UDP-GlcUA (Fig. 1, lane 3). The 37 and 62 kDa bands which photoincorporated [32P]5N3UDP-GlcUA have previously been identified as UDP-glucose:dolichylphosphate glucosyltransferase and phosphoglucomutase, respectively (11). When the same microsomes were photolabeled with [3H]atRA, only one protein band at approximately 52 kDa was significantly photolabeled (Fig. 1, lane 5) and photoincorporation was significantly reduced in the presence of unlabeled atRA. However, in contrast to the results with [32P]5N3UDP-GlcUA, there was some diffuse non-UV dependent photoincorporation into the 50-56 kDa proteins (Fig. 1, lane 4). UDP-hexanolamine-Sepharose affinity chromatography. Solubilized microsomes and concentrated fractions from affinity chromatography were photolabeled with [3H]atRA, proteins were separated by SDS-PAGE
498
AID
BBRC 5992
/
6917$$$$22
12-28-96 07:28:12
bbrcg
AP: BBRC
Vol. 230, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
also were detected on Western blot with the anti-androsterone UGT antibody and the intensity of UGTspecific immunostaining in the fractions paralleled the intensity of photolabeling, except for the flow through fraction (Fig. 2A and 2B, lane 3). The apparent variations in molecular mass of the proteins between samples are probably due to differences in the amounts of protein in the samples: electrophoretic migration was greater in those lanes containing the largest amounts of protein.
FIG. 2. UDP-Hexanolamine Sepharose affinity chromatography of solubilized rat liver microsomal proteins. [3H]atRA photoaffinity labeling and Western blot of solubilized rat liver microsomes and affinity column fractions. (A) Autoradiogram from SDS-PAGE separation of proteins photolabeled with [3H]atRA. (B) Western blot analysis of the same proteins using anti-androsterone-UGT antibody. In both A and B, samples are as follows: lane 1: solubilized microsomes without UV irradiation; lane 2 solubilized microsomes with UV irradiation; lane 3: column flow-through; lane 4: 10-20 ml wash buffer; lane 5: 40-50 ml wash buffer; lane 6: 90-100 ml wash buffer; lane 7: 50 mM UDP-GlcUA eluate.
and the resulting gels were subjected to either autoradiography (Fig. 2A) or Western blot analysis (Fig. 2B). Solubilized microsomes incubated with [3H]atRA without and with UV irradiation (Fig. 2A, lanes 1 and 2) show the same pattern of photoincorporation into 5254 kDa protein bands seen with microsomes (Fig. 1, lane 5). The column flow-through contained a number of photolabeled proteins (Fig. 2A, lane 3) with the most intensely labeled bands being in the 52-54 kDa range while the three wash samples examined (10-20, 4050 and 90-100 ml fractions, Fig. 2A, lanes 4-6) had decreasing amounts of photolabeled protein, almost all of it in the UGT mass range. In the fraction eluted from the column with 50 mM UDP-GlcUA eluate (Fig. 2A, lane 7), only proteins in the UGT weight range were found to photoincorporate [3H]atRA. With Coomassie Blue staining, no significant protein bands outside the UGT range were detected in this fraction. Comparison of the results of photolabeling shown in the autoradiograph in Fig. 2A with the pattern of immunoreactive protein in the same fractions shown in Fig. 2B demonstrates that the two protein bands in the 52-54 kDa range which photoincorporated [3H]atRA
Enzymatic glucuronidation of atRA. Aliquots of rat liver microsomes, solubilized microsomes and affinity column flow through, column wash and 50 mM UDPGlcUA eluate were analyzed for enzymatic glucuronidation activity towards [3H]atRA in order to determine whether active retinoid specific UGT had been recovered from the affinity column. Enzymatic glucuronidation activity in the various fractions is summarized in Table 1. The results demonstrate that full enzymatic activity was recovered after solubilization of microsomal protein and that [3H]atRA glucuronidation activity was 4-5 fold enriched in the UDP-GlcUA eluate from the affinity column as compared to either native or solubilized microsomal protein. DISCUSSION Retinoid glucuronidation has been recognized for some time as a naturally occurring and possibly physiologically relevant biochemical mechanism in rats and humans (23), however, the enzyme catalyzing the reaction has been characterized only as ‘‘a typical microsomal glucuronyl transferase’’ (23). The UGTs are a family of enzymes comprised of multiple isoforms (at least 8 isoforms have been identified in the rat). Thus, there is no ‘‘typical’’ UGT; rather there are a number of enzymes with varying, and sometimes overlapping, substrate specificities. However, all UGTs have two active sites: the UDP-GlcUA site is common to all UGTs, since UDP-GlcUA is the obligatory co-substrate for the glucuronidation reaction, while the aglycone site varies with the substrate specificity of individual isoforms. We have taken advantage of this feature by using pho-
TABLE 1
Enzymatic Glucuronidation of All-trans-Retinoic Acid by Fractions from UDP-Hexanolamine-Sepharose Affinity Chromatography Sample Microsomes Solubilized microsomes Column flow through 50 mM UDPGlcUA eluate
499
AID
BBRC 5992
/
6917$$$$22
12-28-96 07:28:12
bbrcg
AP: BBRC
Activity (pmol/min 1 mg; mean { range) 54.4 58.4 30.6 274.3
{ { { {
1.3 1.7 1.6 3.1
Vol. 230, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
toaffinity probes specific for each site, [32P]5N3UDPGlcUA and [3H]atRA, to determine whether any of the UGT proteins photoincorporate both probes. As can be seen from Fig. 1, at least one protein band of approximately 52 kDa in rat liver microsomes apparently photoincorporated both [32P]5N3UDP-GlcUA (lane 2) and [3H]atRA (lane 5). Labeling of the 50-56 kDa proteins with [32P]5N3UDP-GlcUA was absolutely dependent on UV irradiation and was effectively inhibited (ú80%) by unlabeled UDP-GlcUA. With [3H]atRA, on the other hand, there was some non-UV dependent photolabeling in the range of the UGTs and protection by unlabeled atRA was less effective, with photoincorporation inhibited by approximately 60%. One possible explanation for the non-specific binding is that, due to the long UV exposure time, products of the photolytic degradation of [3H]atRA bind non-specifically to microsomal proteins. An alternative explanation might be provided by direct protein retinoylation, since proteins in several different cell lines (although not microsomes) have been shown to covalently incorporate [3H]atRA (24). Nonetheless, these results indicated that the predominant component of [3H]atRA photolabeling was specific and directed toward the atRA binding site. UDP-Hexanolamine Sepharose affinity chromatography was used to establish that [3H]atRA photolabeled proteins in the 50-56 kDa range were indeed UGTs and not atRA-specific P450s or non-specifically labeled proteins. As can be seen from Fig. 2A, there was significant [3H]atRA photoincorporation into 50-56 kDa proteins in the column flow-through and a decreasing level of photoincorporation was seen in each of the wash fractions. This could be the result of incomplete binding of the UGTs to the resin and/or recovery of proteins, in the same mass range as the UGTs, capable of binding atRA (P450s, RA receptors, retinoylated proteins) but not bound by the affinity resin. Comparison of the pattern of photolabeling seen in Fig. 2A with that of immunoreactive UGT protein in Fig. 2B indicated that both processes contributed, particularly in the case of the flow-through sample. Although the intensity of photolabeling of flow-through proteins was significantly higher than that of solubilized microsomes, the intensity of immunostaining was significantly less, indicating the presence of both UGT protein and non-UGT protein capable of binding [3H]atRA. Elution of the affinity column with UDP-GlcUA resulted in the recovery of two protein bands (52-55 kDa) which were photolabeled by [3H]atRA and recognized by antiUGT antibodies, indicating specific elution of UGT protein. A five-fold enhancement of enzymatic glucuronidation activity toward atRA in this fraction confirmed that active and partially purified UGT protein had been eluted from the column with 50 mM UDP-GlcUA. The results presented here represent the first demonstration of the effectiveness of photoaffinity labeling
with [3H]atRA for identification of retinoid specific UGTs in microsomes. We have shown that UGT isoforms responsible for atRA glucuronidation by rat liver microsomes can be solubilized with full activity and that two isoforms can be at least partially purified by a single affinity chromatographic step. ACKNOWLEDGMENTS This work was supported in part by NIH grants DK45123 and DK49715 to AR.
REFERENCES 1. Dunagin, P. E., Meadows, E. H., Jr., and Olson, J. A. (1965) Science 148, 86–87. 2. Lippel, K., and Olson, J. A. (1968) J. Lipid Res. 9, 580–586. 3. Silva, D. P., Jr., and DeLuca, H. F. (1982) Biochem. J. 206, 33– 41. 4. Barua, A. B., Gunning, D. B., and Olson, J. A. (1991) Biochem. J. 277, 527–531. 5. Lippel, K., and Olson, J. A. (1968) J. Lipid Res. 9, 168–175. 6. Miller, D. A., and DeLuca, H. F. (1986) Arch. Biochem. Biophys. 244, 179–186. 7. Sass, J. O., Forster, A., Bock, K. W., and Nau, H. (1994) Biochemical Pharmacology 47, 485–492. 8. Barua, A. B., and Olson, J. A. (1985) J. Lipid Res. 26, 1277– 1282. 9. Barua, A. B., and Olson, J. A. (1986) Am. J. Clin. Nutr. 43, 481– 485. 10. Drake, R. R., Zimniak, P., Haley, B. E., Lester, R., Elbein, A. D., and Radominska, A. (1991) J. Biol. Chem. 266, 23257–23260. 11. Drake, R., Igari, I., Lester, R., Elbein, A., and Radominska, A. (1992) J. Biol. Chem. 267, 11360–11365. 12. Radominska, A., Berg, C., Treat, S., Little, J. M., Gollan, J., Lester, R., and Drake, R. (1994) Biochim. Biophys. Acta 1195, 63– 70. 13. Radominska, A., Paul, P., Treat, S., Towbin, H., Pratt, C., Little, J., Magdalou, J., Lester, R., and Drake, R. R. (1994) Biochim. Biophys. Acta 1205, 336–345. 14. Little, J. M., Drake, R. R., Vonk, R., Kuipers, F., Lester, R., and Radominska, A. (1995) 273, 1551–1559. 15. Bernstein, P. S., Choi, S.-Y., Ho, Y.-C., and Rando, R. R. (1995) Proc. Nat. Acad. Sci. (USA) 92, 654–658. 16. Radominska, A., and Drake, R. (1994) Methods Enzymol. 230, 330–339. 17. Tukey, R. H., and Tephly, T. R. (1981) Arch. Biochem. Biophys. 209, 565–578. 18. Vanstapel, F., and Blanckaert, N. (1988) Arch. Biochem. Biophys. 263, 216–225. 19. Drake, R. R., Evans, R. K., Wolf, M. J., and Haley, B. E. (1989) J. Biol. Chem. 264, 11928–11933. 20. Laemmli, U. K. (1970) Nature 227, 680–685. 21. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350–4354. 22. Little, J. M., Lehman, P. A., Nowell, S., Samokyszyn, V., and Radominska, A. Drug Metab. Disp., in press. 23. Blaner, W. S., and Olson, J. A. (1994) in The Retinoids. Biology, Chemistry, and Medicine (M. B. Sporn, A. B. Roberts, and D. S. Goodman, Eds.), pp. 229–255, Raven Press, New York. 24. Breitman, T. R., and Takahashi, N. (1996) Biochemical Society Transactions 24, 723–727.
500
AID
BBRC 5992
/
6917$$$$22
12-28-96 07:28:12
bbrcg
AP: BBRC
230, 497–500 (1997)
RC965992
Application of Photoaffinity Labeling with [11,12-3H]Alltrans-Retinoic Acid to Characterization of Rat Liver Microsomal UDP-Glucuronosyltransferase(s) with Activity toward Retinoic Acid Joanna M. Little1 and Anna Radominska Division of Gastroenterology, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Received December 9, 1996
[3H]All-trans-retinoic acid has been shown to be an effective photoaffinity label for microsomal UDP-glucuronosyltransferases. Labeling of rat liver microsomal proteins with [3H]all-trans-retinoic acid and [32P]5-azido-UDP-glucuronic acid has shown that at least one protein in the 50-56 kDa mass range encompassing the UDP-glucuronosyltransferases photoincorporated both probes. The fraction of solubilized microsomal protein eluted from a UDP-hexanolamine affinity column with 50 mM UDP-glucuronic acid contained two protein bands, both of which photoincorporated [3H]alltrans-retinoic acid and were detected on Western blot by anti-UDP-glucuronosyltransferase antibodies. Enzymatic glucuronidation activity toward atRA in the same fraction was enriched five-fold over that of native or solubilized microsomes. q 1997 Academic Press
Glucuronidation is generally considered to be a reaction of detoxification and elimination. UDP-glucuronosyltransferases (UGTs, EC 2.4.1.17), the enzymes responsible for the glucuronidation reaction, have also been found to play a powerful role in the metabolism of endogenous substances as well as the detoxification of drugs and other xenobiotics. Retinoic acid, a physiologically and pharmacologically important compound, can be metabolized and excreted in the form of an acyl glucuronide. Retinoyl-b-glucuronide was first identi1 To whom correspondence should be addressed at Division of Gastroenterology, University of Arkansas for Medical Science, 4301 W. Markham, Slot 567, Little Rock, AR 72205. Fax: 501-686-6248. Abbreviations: UGT: UDP-glucuronosyltransferase; atRA: all trans-retinoic acid; atRAG: all trans retinoyl-b-glucuronide; [32P]5N3UDP-GlcUA: [b-32P]5-azido-UDP-glucuronic acid; UDP-GlcUA: UDP-glucuronic acid; SDS-PAGE: SDS-polyacrylamide gel electrophoresis; DTT: dithiothreitol; PC: phosphatidyl choline; DTT: dithiothreitol.
fied thirty years ago as a naturally occurring product of the in vivo metabolism of atRA in rats (1). Since that time, in vivo glucuronidation of atRA administered to rats has been confirmed (2-4), in vitro glucuronidation of atRA has been demonstrated in rat liver microsomes (5-7) and rat kidney and intestinal microsomes as well (5) and [11-3H]atRAG has been chemically synthesized and its in vivo metabolism in the rat has been studied (8). In addition, atRAG has been shown to be an endogenous component of human blood (9). This study was prompted by the fact that scant attention has been paid to the characterization or identification of the UGT(s) responsible for the conjugation reaction. We have applied [32P]5N3UDP-GlcUA photoaffinity labeling, which has proven to be very effective in characterizing rat and human liver microsomal UGTs as a group (10-14) and the recently published method of Bernstein et al (15) for direct photoaffinity labeling with [11,12-3H]atRA to the identification of the UGT(s) in rat liver microsomes responsible for RA glucuronidation. Experiments have shown that at least one rat liver microsomal protein in the 50-56 kDa molecular mass range known to contain the UGTs can be photolabeled with both [3H]atRA, a probe for the aglycone binding site, and [32P]5N3UDP-GlcUA, a UDP-GlcUA binding site directed photoaffinity analog. Additionally, two protein bands in the 50-56 kDa range recovered in fractions eluted from a UDP-affinity column with UDPGlcUA were found to photoincorporate [3H]atRA and to be detected by an anti-UGT antibody on Western blots. Enzymatic glucuronidation of RA by these same fractions was found to be 5-fold enriched over that of the solubilized microsomal proteins. MATERIALS AND METHODS Materials. [11,12-3H]atRA was purchased from Dupont-New England Nuclear (Boston, MA). [32P]5N3-UDP-GlcUA was synthesized
497
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 5992
/
6917$$$$21
12-28-96 07:28:12
bbrcg
AP: BBRC
Vol. 230, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and purified as previously described (10, 16). [32P]Pi was from ICN (Costa Mesa, CA). Brij 58, UDP-GlcUA, unlabeled atRA and saccharolactone were purchased from Sigma Chemical Co. (St. Louis, MO). UDP-hexanolamine (Sigma) was linked to cyanogen bromide activated Sepharose 4B (Sigma) as previously described (17). Microsome preparation and solubilization. Male Sprague-Dawley rats (200-250 g) were used and microsomes prepared as described in (18), except that they were not further subfractionated into rough and smooth ER fractions. For solubilization of membrane proteins, microsomes as prepared above were suspended in 10 mM Tris-HCl, pH 7.5 containing 0.5 M KCl, 0.05 mg/ml PC (from egg yolk, Sigma) and 10% glycerol (w/v) at a protein concentration of 3-5 mg/ml. CHAPS was added to a final concentration of 4 mM and the suspension was incubated on ice, with stirring, for one hour followed by centrifugation at 105,000 1 g for one hour. The supernatant was removed and stored at 0207C until use. All protein determinations were carried out using the BioRad protein assay kit (BioRad, La Jolla, CA) with bovine serum albumin as standard. Photoaffinity labeling. The standard protocol for photolabeling of rat liver microsomes with [32P]5N3UDP-GlcUA has been described in detail (11-13, 19). Photolabeling with [11,12-3H]RA was done using the method of Bernstein et al (15) modified as follows. For studies with detergent activated microsomes, [3H]RA (solubilized in micellar form with Triton-X100) was added to rat liver microsomal protein (100 mg) in 100 mM HEPES, pH 7.0 containing 5 mM MgCl2 to a final concentration of 2 mM (2.0 mCi) [3H]RA and 0.05% Triton in a total volume of 25 ml. The reaction was incubated on ice for 10 min followed by irradiation with a hand-held long-wave UV lamp (366 nm, UVP-21, Ultraviolet Products, Inc., San Gabriel, CA) for 15 min on ice. All experiments involving RA were carried out under yellow light. All photolabeling experiments were stopped with 10% TCA (150 ml) and processed for SDS-PAGE on 10% gels (20) as previously described (19). Gels were stained, dried and subjected to autoradiography for 1-7 days. Gels with proteins labeled with [3H]RA were treated with Autofluor (National Diagnostics, Manville, NJ) according to the manufacturer’s directions prior to drying. Autoradiographs were analyzed by densitometry with a BioRad Imaging Densitometer (BioRad, La Jolla, CA). In some cases, proteins were electroblotted from gels to nitrocellulose and subjected to Western blot analysis using the method of Towbin et al (21). Blotted proteins were probed with a rat anti-androsterone UGT antibody, a generous gift from M. Green and Dr. T. Tephly, University of Iowa, Iowa City, IA. UDP-hexanolamine-Sepharose affinity chromatography. All of the following manipulations were carried out at 47C. UDP-hexanolamine-Sepharose was equilibrated with 20 mM HEPES, pH 6.5 containing 10% glycerol, 0.05% CHAPS, 0.1 mM DTT and 25 mM KCl. Solubilized rat liver microsomal protein (approximately 2 mg, prepared as described above) was diluted with 5 ml of equilibration buffer supplemented with 5 mM MgCl2 and 0.05 mg/ml PC (Buffer B). This sample was mixed with 1 ml of UDP-hexanolamine-Sepharose in a small column (BioRad Poly-Prep Chromatography Column), both ends of the column were sealed, and sample and resin were gently shaken overnight. After the resin had been allowed to settle in the column, the sample was allowed to flow through the column under gravity at a flow rate not exceeding 0.5 ml/min. The effluent was collected as a single fraction (flow-through). The column was washed with 50 ml Buffer B, collecting 5 ml fractions, and protein bound to the column was eluted with 5 ml of 50 mM in Buffer B, collected as a single fraction. Fractions were concentrated using Centricon 30 ultrafilters (Amicon, Beverly, MA). Duplicate aliquots of microsomal proteins, solubilized microsomes and concentrated fractions were photolabeled with [3H]atRA as described above and subjected to SDS-PAGE. One gel was processed for autoradiography and the other for Western blotting as described above. Additional aliquots were assayed for enzymatic glucuronidation activity towards RA as described below.
FIG. 1. Photoaffinity labeling of rat liver microsomes with 40 mM [32P]5N3UDP-GlcUA or 2 mM [3H]atRA. Lanes 1-3: photolabeling with [32P]5N3UDP-GlcUA; lane 1: no UV irradiation; lane 2: UV irradiation; lane 3: UV irradiation, with 0.4 mM UDP-GlcUA. Lanes 46: photolabeling with [3H]atRA; lane 4: no UV irradiation; lane 5: UV irradiation; lane 6: UV irradiation, with 20 mM atRA.
Enzymatic assays. Enzymatic retinoid glucuronidation activity in rat liver microsomes was determined as previously described (22). The detergent Brij-58 (final concentration, 0.05%) was used both to solubilize the hydrophobic substrate in micellar form and to activate the enzymatic activity of the microsomal UGTs. All manipulations were carried out under yellow light.
RESULTS Photoaffinity labeling of rat liver microsomes with [32P]5N3UDP-GlcUA and [3H]atRA. The autoradiograph in Figure 1 shows the results of a representative experiment in which rat liver microsomes were photolabeled with either [32P]5N3UDP-GlcUA (lanes 1-3) or [3H]atRA (lanes 4-6) followed by SDS-PAGE separation of microsomal proteins. At least two protein bands (approximately 52 and 54 kDa) in the 50-56 kDa range known to encompass the UGTs (10) were photolabeled with [32P]5N3UDP-GlcUA only after UV irradiation (compare Fig. 1, lanes 1 and 2, without and with UV irradiation, respectively) and the photoincorporation was effectively inhibited by unlabeled UDP-GlcUA (Fig. 1, lane 3). The 37 and 62 kDa bands which photoincorporated [32P]5N3UDP-GlcUA have previously been identified as UDP-glucose:dolichylphosphate glucosyltransferase and phosphoglucomutase, respectively (11). When the same microsomes were photolabeled with [3H]atRA, only one protein band at approximately 52 kDa was significantly photolabeled (Fig. 1, lane 5) and photoincorporation was significantly reduced in the presence of unlabeled atRA. However, in contrast to the results with [32P]5N3UDP-GlcUA, there was some diffuse non-UV dependent photoincorporation into the 50-56 kDa proteins (Fig. 1, lane 4). UDP-hexanolamine-Sepharose affinity chromatography. Solubilized microsomes and concentrated fractions from affinity chromatography were photolabeled with [3H]atRA, proteins were separated by SDS-PAGE
498
AID
BBRC 5992
/
6917$$$$22
12-28-96 07:28:12
bbrcg
AP: BBRC
Vol. 230, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
also were detected on Western blot with the anti-androsterone UGT antibody and the intensity of UGTspecific immunostaining in the fractions paralleled the intensity of photolabeling, except for the flow through fraction (Fig. 2A and 2B, lane 3). The apparent variations in molecular mass of the proteins between samples are probably due to differences in the amounts of protein in the samples: electrophoretic migration was greater in those lanes containing the largest amounts of protein.
FIG. 2. UDP-Hexanolamine Sepharose affinity chromatography of solubilized rat liver microsomal proteins. [3H]atRA photoaffinity labeling and Western blot of solubilized rat liver microsomes and affinity column fractions. (A) Autoradiogram from SDS-PAGE separation of proteins photolabeled with [3H]atRA. (B) Western blot analysis of the same proteins using anti-androsterone-UGT antibody. In both A and B, samples are as follows: lane 1: solubilized microsomes without UV irradiation; lane 2 solubilized microsomes with UV irradiation; lane 3: column flow-through; lane 4: 10-20 ml wash buffer; lane 5: 40-50 ml wash buffer; lane 6: 90-100 ml wash buffer; lane 7: 50 mM UDP-GlcUA eluate.
and the resulting gels were subjected to either autoradiography (Fig. 2A) or Western blot analysis (Fig. 2B). Solubilized microsomes incubated with [3H]atRA without and with UV irradiation (Fig. 2A, lanes 1 and 2) show the same pattern of photoincorporation into 5254 kDa protein bands seen with microsomes (Fig. 1, lane 5). The column flow-through contained a number of photolabeled proteins (Fig. 2A, lane 3) with the most intensely labeled bands being in the 52-54 kDa range while the three wash samples examined (10-20, 4050 and 90-100 ml fractions, Fig. 2A, lanes 4-6) had decreasing amounts of photolabeled protein, almost all of it in the UGT mass range. In the fraction eluted from the column with 50 mM UDP-GlcUA eluate (Fig. 2A, lane 7), only proteins in the UGT weight range were found to photoincorporate [3H]atRA. With Coomassie Blue staining, no significant protein bands outside the UGT range were detected in this fraction. Comparison of the results of photolabeling shown in the autoradiograph in Fig. 2A with the pattern of immunoreactive protein in the same fractions shown in Fig. 2B demonstrates that the two protein bands in the 52-54 kDa range which photoincorporated [3H]atRA
Enzymatic glucuronidation of atRA. Aliquots of rat liver microsomes, solubilized microsomes and affinity column flow through, column wash and 50 mM UDPGlcUA eluate were analyzed for enzymatic glucuronidation activity towards [3H]atRA in order to determine whether active retinoid specific UGT had been recovered from the affinity column. Enzymatic glucuronidation activity in the various fractions is summarized in Table 1. The results demonstrate that full enzymatic activity was recovered after solubilization of microsomal protein and that [3H]atRA glucuronidation activity was 4-5 fold enriched in the UDP-GlcUA eluate from the affinity column as compared to either native or solubilized microsomal protein. DISCUSSION Retinoid glucuronidation has been recognized for some time as a naturally occurring and possibly physiologically relevant biochemical mechanism in rats and humans (23), however, the enzyme catalyzing the reaction has been characterized only as ‘‘a typical microsomal glucuronyl transferase’’ (23). The UGTs are a family of enzymes comprised of multiple isoforms (at least 8 isoforms have been identified in the rat). Thus, there is no ‘‘typical’’ UGT; rather there are a number of enzymes with varying, and sometimes overlapping, substrate specificities. However, all UGTs have two active sites: the UDP-GlcUA site is common to all UGTs, since UDP-GlcUA is the obligatory co-substrate for the glucuronidation reaction, while the aglycone site varies with the substrate specificity of individual isoforms. We have taken advantage of this feature by using pho-
TABLE 1
Enzymatic Glucuronidation of All-trans-Retinoic Acid by Fractions from UDP-Hexanolamine-Sepharose Affinity Chromatography Sample Microsomes Solubilized microsomes Column flow through 50 mM UDPGlcUA eluate
499
AID
BBRC 5992
/
6917$$$$22
12-28-96 07:28:12
bbrcg
AP: BBRC
Activity (pmol/min 1 mg; mean { range) 54.4 58.4 30.6 274.3
{ { { {
1.3 1.7 1.6 3.1
Vol. 230, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
toaffinity probes specific for each site, [32P]5N3UDPGlcUA and [3H]atRA, to determine whether any of the UGT proteins photoincorporate both probes. As can be seen from Fig. 1, at least one protein band of approximately 52 kDa in rat liver microsomes apparently photoincorporated both [32P]5N3UDP-GlcUA (lane 2) and [3H]atRA (lane 5). Labeling of the 50-56 kDa proteins with [32P]5N3UDP-GlcUA was absolutely dependent on UV irradiation and was effectively inhibited (ú80%) by unlabeled UDP-GlcUA. With [3H]atRA, on the other hand, there was some non-UV dependent photolabeling in the range of the UGTs and protection by unlabeled atRA was less effective, with photoincorporation inhibited by approximately 60%. One possible explanation for the non-specific binding is that, due to the long UV exposure time, products of the photolytic degradation of [3H]atRA bind non-specifically to microsomal proteins. An alternative explanation might be provided by direct protein retinoylation, since proteins in several different cell lines (although not microsomes) have been shown to covalently incorporate [3H]atRA (24). Nonetheless, these results indicated that the predominant component of [3H]atRA photolabeling was specific and directed toward the atRA binding site. UDP-Hexanolamine Sepharose affinity chromatography was used to establish that [3H]atRA photolabeled proteins in the 50-56 kDa range were indeed UGTs and not atRA-specific P450s or non-specifically labeled proteins. As can be seen from Fig. 2A, there was significant [3H]atRA photoincorporation into 50-56 kDa proteins in the column flow-through and a decreasing level of photoincorporation was seen in each of the wash fractions. This could be the result of incomplete binding of the UGTs to the resin and/or recovery of proteins, in the same mass range as the UGTs, capable of binding atRA (P450s, RA receptors, retinoylated proteins) but not bound by the affinity resin. Comparison of the pattern of photolabeling seen in Fig. 2A with that of immunoreactive UGT protein in Fig. 2B indicated that both processes contributed, particularly in the case of the flow-through sample. Although the intensity of photolabeling of flow-through proteins was significantly higher than that of solubilized microsomes, the intensity of immunostaining was significantly less, indicating the presence of both UGT protein and non-UGT protein capable of binding [3H]atRA. Elution of the affinity column with UDP-GlcUA resulted in the recovery of two protein bands (52-55 kDa) which were photolabeled by [3H]atRA and recognized by antiUGT antibodies, indicating specific elution of UGT protein. A five-fold enhancement of enzymatic glucuronidation activity toward atRA in this fraction confirmed that active and partially purified UGT protein had been eluted from the column with 50 mM UDP-GlcUA. The results presented here represent the first demonstration of the effectiveness of photoaffinity labeling
with [3H]atRA for identification of retinoid specific UGTs in microsomes. We have shown that UGT isoforms responsible for atRA glucuronidation by rat liver microsomes can be solubilized with full activity and that two isoforms can be at least partially purified by a single affinity chromatographic step. ACKNOWLEDGMENTS This work was supported in part by NIH grants DK45123 and DK49715 to AR.
REFERENCES 1. Dunagin, P. E., Meadows, E. H., Jr., and Olson, J. A. (1965) Science 148, 86–87. 2. Lippel, K., and Olson, J. A. (1968) J. Lipid Res. 9, 580–586. 3. Silva, D. P., Jr., and DeLuca, H. F. (1982) Biochem. J. 206, 33– 41. 4. Barua, A. B., Gunning, D. B., and Olson, J. A. (1991) Biochem. J. 277, 527–531. 5. Lippel, K., and Olson, J. A. (1968) J. Lipid Res. 9, 168–175. 6. Miller, D. A., and DeLuca, H. F. (1986) Arch. Biochem. Biophys. 244, 179–186. 7. Sass, J. O., Forster, A., Bock, K. W., and Nau, H. (1994) Biochemical Pharmacology 47, 485–492. 8. Barua, A. B., and Olson, J. A. (1985) J. Lipid Res. 26, 1277– 1282. 9. Barua, A. B., and Olson, J. A. (1986) Am. J. Clin. Nutr. 43, 481– 485. 10. Drake, R. R., Zimniak, P., Haley, B. E., Lester, R., Elbein, A. D., and Radominska, A. (1991) J. Biol. Chem. 266, 23257–23260. 11. Drake, R., Igari, I., Lester, R., Elbein, A., and Radominska, A. (1992) J. Biol. Chem. 267, 11360–11365. 12. Radominska, A., Berg, C., Treat, S., Little, J. M., Gollan, J., Lester, R., and Drake, R. (1994) Biochim. Biophys. Acta 1195, 63– 70. 13. Radominska, A., Paul, P., Treat, S., Towbin, H., Pratt, C., Little, J., Magdalou, J., Lester, R., and Drake, R. R. (1994) Biochim. Biophys. Acta 1205, 336–345. 14. Little, J. M., Drake, R. R., Vonk, R., Kuipers, F., Lester, R., and Radominska, A. (1995) 273, 1551–1559. 15. Bernstein, P. S., Choi, S.-Y., Ho, Y.-C., and Rando, R. R. (1995) Proc. Nat. Acad. Sci. (USA) 92, 654–658. 16. Radominska, A., and Drake, R. (1994) Methods Enzymol. 230, 330–339. 17. Tukey, R. H., and Tephly, T. R. (1981) Arch. Biochem. Biophys. 209, 565–578. 18. Vanstapel, F., and Blanckaert, N. (1988) Arch. Biochem. Biophys. 263, 216–225. 19. Drake, R. R., Evans, R. K., Wolf, M. J., and Haley, B. E. (1989) J. Biol. Chem. 264, 11928–11933. 20. Laemmli, U. K. (1970) Nature 227, 680–685. 21. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350–4354. 22. Little, J. M., Lehman, P. A., Nowell, S., Samokyszyn, V., and Radominska, A. Drug Metab. Disp., in press. 23. Blaner, W. S., and Olson, J. A. (1994) in The Retinoids. Biology, Chemistry, and Medicine (M. B. Sporn, A. B. Roberts, and D. S. Goodman, Eds.), pp. 229–255, Raven Press, New York. 24. Breitman, T. R., and Takahashi, N. (1996) Biochemical Society Transactions 24, 723–727.
500
AID
BBRC 5992
/
6917$$$$22
12-28-96 07:28:12
bbrcg
AP: BBRC