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Preparation and characterization of radioactive castanospermine
ANALYTICAL
BIOCHEMISTRY
Preparation
163,3 16-32 1 (I 987)
and Characterization ROY W. KEENAN,~.
of Radioactive T. PAN,ANDALAND.
Castanospermine’ ELBEIN
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284 Received October 2 1, 1986 A procedure for the preparation of tritiated castanospennine is described. The tritiated alkaloid was shown to be chromatographically identical to the native material and exhibited the same inhibitory properties. Radiolabeled castanospermine tightly bound to purified intestinal sucrase. Following gel chromatography, each mole of enzyme was shown to have bound 1 mol of the radioactive alkaloid. Cultured MDCK cells were also shown to take up the labeled castanospermine. This compound should be a useful tool in the investigation of enzymes that are responsible for the processing of glycoprotein oligosaccharides. o 1987 Academic R.ZSS, IIIC. KEY WORDS: castanospermine; tritium labeling; amyloglucosidase; processing enzymes.
Castanospermine [( lS,&S,7R,SR,8aR)1,6,7,&tetrahydroxyindolizidine] is an indolizidine alkaloid that was isolated from the seeds of the Australian legume, Castanospermum australe (1). Animals that eat these seeds develop severe gastrointestinal symptoms that may ultimately lead to death (2). These symptoms are probably due to the fact that the alkaloid is a potent inhibitor of intestinal sucrase and maltase, as well as of other cu-glucosidases (3). In addition, when injected into rats this alkaloid strongly inhibits cY-glucosidase activity in a variety of tissues, notably the liver. After daily injections of the alkaloid for 3 days, there is an almost complete absence of glycogen granules from the cytoplasm of hepatocytes, and instead the glycogen accumulates in the lysosomes (4). Thus, in these animals, castanospermine appears to cause symptoms like those of human Pompe’s disease. In addition, castanospermine inhibits the processing of the oligosaccharide chains of the N-linked glycoproteins by virtue of the fact that it inhibits glucosidase I (and also glucosidase II) (5). As a result, cells are not able to produce ’ This investigation was supported by NIH Grants 17897-l 1 to Roy W. Keenan and AM 2 1800 and GM 3 1355 to Alan D. Elbein. 0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
the typical complex chains, and their glycoproteins instead have GlcsMan7-9(GlcNAc)2 structures* (6-9). Since the alkaloid has such interesting and potentially important effects on a variety of systems, it would be of considerable value to have radioactive castanospermine in order to be able to trace its location in cells and in animals, as well as its possible metabolism. This manuscript describes the preparation of 3H-labeled castanospermine and some of the effects of this labeled alkaloid on biological systems. MATERIALS
AND METHODS
Materials. Tritiated water (1 Ci/g) was obtained from DuPont/New England Nuclear Co. and deuterium oxide (99.96% atom excess) from Aldrich Chemical Co. Castanospermine was isolated and purified from the seeds of C. australe (2). Sucrase was isolated from rat intestine and purified to homogeneity (3). All solvents and chemicals either were the best grade available or were specially purified for HPLC. Methods. Mass spectral analysis was performed by Dr. Susan Weintraub, Depart2 Abbreviations used: GlcNAc, N-acetylglucosamine; SDS, sodium dodecyl sulfate. 316
CHROMATOGRAPHIC
PREPARATION
OF RADIOACTIVE
CASTANOSPERMINE
317
ment of Pathology, on a Hewlett-Packard lute acetic acid and applied to a 0.5 X 7-cm Model 5982 mass spectrometer in combinacolumn of Dowex 50-8X NH:. The resin tion with a Hewlett-Packard Model 5933 was washed with 7 ml of water, followed by data system. Samples were introduced by elution with 50 ml of a 0 to 0.1 M NH40H means of a direct insertion probe and were gradient. Approximately 50% of the applied analyzed by electron impact at 70 meV with activity eluted in one peak as shown in Fig. 1. an ion source temperature of 180°C. VolatilApproximately equal portions of the reization of the castanospermine occurred at maining activity were eluted in the water approximately 84°C. wash or remained bound to the column. The The ‘H NMR spectra were run on a Gen- overall yield of radioactive castanospermine eral Electric QE-300 NMR spectrophotomewas estimated to be 50% based on its inhibiter by Dr. Robert Williams and Tony Her- tion of cu-glucosidase activity, and the spenandez, Chemistry Department, University cific activity was estimated to be 25.8 mCi/ of Texas (San Antonio, TX). mmol. Samples of the radioactive compound Castanospermine was analyzed by highwere incubated for 48 h at 50°C in either pressure liquid chromatography on a Waters 0.0 1 N HCl or 0.0 1 N NaOH. HPLC analysis radial compression reversed-phase column of the products of these incubations revealed (ODS 18, 10 pm). The chromatography sol- that there was no exchange of tritium with vent was 0.0 1 M sodium octanesulfonate (pH the solvent in either case, showing that the 3.8):acetonitrile (10: 1, v/v) which was run at label is sufficiently stable to use in biological 1 ml/min. Purified radioactive castanosperexperiments. mine eluted as a single peak of radioactivity RESULTS AND DISCUSSION after approximately 10 min. Radioactivity The Purification and Characterization of was determined with a flowthrough detector Radioactive Castanospermine (Flo-One-B Model 1C radioactive detector) in most chromatographic runs. A small quantity of tritiated castanosperPreparation of [3H]castanospermine. Cas- mine, prepared by exchange in tritiated tanospermine was labeled by exchange with tritiated water in the presence of Adams catalyst (10). Platinum oxide was activated by reacting with ethanolic NaBH4. An aliquot of the suspended catalyst containing approximately 1.8 mg was added to a 0.5-ml conical vial and most of the solvent was removed under a stream of nitrogen. Tritiated water (75 ~1, 75 mCi) and 6.1 mg of castanospermine were added and the Teflon-capped vial was placed in a 125 “C heating block for 22 h. Following incubation, the cooled sample was transferred to an Eppendorf centrifuge tube together with several washings. The catalyst FRACTION NUMBER was sedimented by centrifugation, and the FIG. 1. Isolation of [3H]castanospermine by ion-exsupernatant was removed. The catalyst was change chromatography. Tritiated castanospermine washed several additional times and the su- prepared as described under Materials and Methods was applied to a 7 X 70-mm column of Dowex 50 NH; and pernatants were combined. The combined extracts were acidified with acetic acid, and eluted with approximately 10 ml of water. A linear gradient of NH,OH (25 ml water in the mixing vessel and tritiated water was largely removed by lyo- 0.1 M NH,OH in the reservoir) was begun at tube 1. philizing the sample in a microlyophilization Fractions (0.7 I ml) were collected and 15-4 aliquots apparatus. The residue was dissolved in di- were assayed for radioactivity.
318
KEENAN,
PAN, AND ELBEIN
water as described under Materials and Methods, was mixed with carrier castanospermine and subjected to HPLC. Aliquots of the chromatographic eluate were analyzed for both radioactivity and ability to inhibit amyloglucosidase. Figure 2 shows that there was good correlation between the elution profile of the radioactivity and that of the inhibitory activity. In order to further characterize the product, castanospermine was carried through the same procedure on a threefold larger scale in the presence of 100% deuterium oxide. The mass spectrum of the deuterated product revealed a mixture of different products containing from one to seven deuterium atoms. The major peaks showed two to four deuterium atoms incorporated, with the largest peak being that with three protons replaced. A comparison of the NMR spectra of the deuterated sample and the starting material revealed the presence of several peaks in the deuterated samples that were not in the starting material. These were removed when the samples were purified by ion-exchange chromatography. A comparison of the NMR spectra of the starting material and of the
FIG. 2. HPLC oftritiated castanospermine. An aliquot of tritium-labeled castanospermine was mixed with 2.4 nmol of nonlabeled castanospermine and subjected to reversed-phase HPLC. Fractions (0.3 ml) were collected and analyzed for radioactivity and for inhibitory activity against cy-glucosidase. The chromatography conditions and assays were as described under Materials and Methods.
1~~~~1’~“1’~~‘(~“~1~~‘~1~“~1 4.5 4.0 3.5
3.0
2.5
2.0
1.5
PPM
B
FIG. 3. NMR spectra of castanospermine. (A) Castanospermine; (B) castanospermine incubated with deuterium oxide under conditions identical to those used to incorporate tritium. Both spectra were taken in deuterium oxide. Details of the procedure and NMR assayare given under Materials and Methods.
purified deuterated product revealed that a number of protons had been replaced. Using the assignments provided by Hohenschultz et al. (1) it was determined that the protons on the carbons bearing the hydroxyl groups did not undergo exchange. Although precise assignment could not be made in all cases, there is no doubt that most of the exchange took place with protons (Y to the tertiary amine group (carbons 3 and 5, and the methine carbon at the ring juncture; see Fig. 3). Some exchange also took place at carbon 2, but not to the extent seen with the carbons (Yto the nitrogen. It seems likely that the purified radiolabeled castanospermine is the same stereoisomer as the starting material because the compound appeared to be chromatographitally homogeneous on both ion-exchange chromatography and HPLC. A mixture of epimers would probably show different chromatographic behavior. Also, changes in the stereochemistry of the molecule should produce changes in its activity as an inhibitor; however, these were not observed. A more compelling piece of evidence against the formation of an epimeric mixture is that the NMR spectrum (Fig. 3) provides evidence
CHROMATOGRAPHIC
PREPARATION
that no deuterium exchange on carbons 1,6, 7, and 8 occurred when the reaction was carried out under the same conditions in deuterated water. Mechanisms of epimerization involving a carbonium ion or olefin would be expected to result in deuterium incorporation into these positions.
Fate of Injected Radioactive Castanospermine Tritiated castanospermine was injected into the tail vein of a 200-g female rat (2.65 nmol/g). After 24 h, the levels of radioactivity in tissue and those in the feces and urine were determined. Approximately 80% of the injected dose was recovered in the urine and cage washings. Liver, brain, pancreas, heart, lung, kidney, spleen, adrenals, ovaries, and intestine were assayed for radioactivity. Although there were some differences in the relative amounts of radioactivity in these tissues, none of the tissues contained over a fraction of a percent of the injected radioactivity. The results obtained in this experiment were typical of those in two other experiments in which rats were injected with labeled castanospermine. All the radioactivity in the urine was shown to cochromatograph with authentic samples of castanospermine. Thus, it is clear that castanospermine is absorbed very poorly by tissues in the rat, is not metabolized to any significant extent, and is rapidly excreted in the urine. In earlier investigations in which this alkaloid was shown to have an effect on liver glycosidases, much larger doses were administered over a period of several days (4).
Biological Activity of the Labeled Castanospermine The labeled castanospermine was compared to authentic castanospermine in terms of its ability to inhibit purified rat intestinal sucrase. Figure 4 shows inhibition curves comparing the effects of increasing amounts of the two castanospermine preparations against the sucrase. It can be seen that both preparations were potent inhibitors of this
OF RADIOACTIVE
319
CASTANOSPERMINE
enzyme and the inhibition curves were almost identical. Thus, in both cases, 50% inhibition occurred at about 25 to 30 rig/ml. Since one of the advantages of having labeled castanospermine would be to localize a-glucosidases within cells or tissues, we examined the binding of [3H]castanospermine to purified intestinal sucrase to determine whether the radioactivity would remain with the enzyme and how much could be bound. Various amounts of sucrase were mixed with 100 pg of labeled castanospermine and the mixture was applied to a column of Sephadex G-200. Fractions eluting from the column were assayed for radioactivity and for enzyme activity. Figure 5 shows that a symmetrical peak of radioactivity emerged from the column in the same fractions as the enzymatic activity. The height of the radioactive peak (i.e., the amount of radioactivity bound) increased in direct proportion to the amount of sucrase used. In addition, a relatively large amount of radioactivity was retained on the column and emerged in fractions 130 and beyond. These are the fractions where low-molecular-weight material elutes, and this radioactivity represents un-
-. 0
I 50 INHIBITOR
I I 100 150 CONCENTRATION
1 200
I
(nghnl)
FIG. 4. Inhibition of sucrose activity by [‘Hlcastanospermine. Purified sucrase was assayed for its ability to cleave sucrose in the presence of various amounts of authentic castanospermine or [3H]castanospermine. Reactions were initiated by the addition of sucrose, and following an incubation of 10 mitt, the amount of reducing sugar released was measured at 520 mM by Nelson’s assay.
320
KEENAN,
PAN, AND ELBEIN
bound castanospermine. A calculation of the extent of binding using the data from Fig. 5 revealed that exactly 1 mol of castanospermine was bound per mole of sucrase. An additional experiment identical to the one shown in Fig. 5 was performed, except that the enzyme was incubated with labeled castanospermine in the presence of a 43-fold excess of unlabeled castanospermine. The result of this experiment (not shown) was that the amount of enzyme-bound radioactivity was decreased to the calculated level. This is further evidence that the radioactive castanospermine has the same biological properties as the unlabeled material. We also examined the binding, or uptake, of castanospermine by MDCK cells growing on plastic dishes. In these experiments confluent monolayers were incubated with various amounts of [3H]castanospermine for 30 min. The monolayers were then washed four or five times with phosphate-buffered saline to remove the free castanospermine. The cells were released from the plates by adding 1% SDS, and the cell suspensions were counted to determine the amount of radioactivity. Figure 6 shows that [3H]castanospermine was taken up by the cells in a dose-de-
SUCRASE 4
NUMBER
FIG. 5. Binding of [3H]castanospermine to purified sucrase. Various amounts of homogeneous enzyme were mixed with 100 fig of [‘Hlcastanospermine in 0.01 M sodium phosphate buffer, pH 7.5, in a final volume of 0.6 ml as indicated in the figure. The mixture was applied to a 1.5 X lOO-cm column of Sephadex G-200. Fractions eluting from the column were assayed for radioactivity and for sucrase activity.
I
I
I
120 160 80 OF CASTANOSPERMINE
I
200 (pglml)
6. Binding of [‘Hlcastanospermine to MDCK cells. Confluent monolayers of MDCK cells were incubated with various amounts of [3H]castanospermine for 30 min in modified Eagle’s medium containing 2% fetal calf serum as indicated in the figure. The monolayers were washed thoroughly with phosphate-buffered saline and the cells were released from the plates by adding I % SDS. The cell suspensions were counted to determine the amount of ‘H bound. FIG.
pendent manner and that the radioactivity does appear to enter the cells. However, the uptake of castanospermine by the MDCK cells may not be carrier mediated, since it was not possible to block this uptake by adding unlabeled castanospermine or swainsonine; or, it is possible that the amount of unlabeled castanospermine added was not enough to dilute the label. At any rate, the fact that castanospermine is taken up by these cells should make it possible to localize the alkaloid in various cell fractions. Castanospermine is a toxic plant alkaloid that has potent effects on various biological systems, including the inhibition of processing of N-linked oligosaccharides. In order to understand how this alkaloid affects cells in culture or animals, it would be valuable to have radioactive castanospermine. This would allow one to localize castanospermine within the cell or to determine its tissue distribution and also to determine whether this compound is metabolized in animals. Since castanospermine is found only in the seeds of the Australian tree C. austrule it does not appear feasible to label this alkaloid in vivo. Also, since nothing is known about the biosynthetic pathway of this compound, it is not possible at this point to isolate or label inter-
1 14
FRACTION
I
40 AMOUNT
CHROMATOGRAPHIC
PREPARATION
mediates. Therefore, we have developed a method to label the alkaloid with tritium by an exchange method. Since the label is stable, the compound can be used for a variety of biological studies. Some biological studies with the [3H]castanospermine are demonstrated in this report. REFERENCES 1. Hohenschultz, L. D., Bell, E. A., Jewess, P. P., Leworthy, D. P., Pryce. R. J., Arnold, E., and Clardy, J. (198 1) Phyfochemistry 20, 8 1 l-8 14. 2. Nash, R. J., Evans, S. V., Fellows, L. E., and Bell, E. A. (1985) in Plant Toxicology (Seawright, A. A., Hegarty, M. P., James, L. F., and Keeler, R. F., Ed%), pp. 309-3 14, Queensland Poisonous Plant Committee, Yeerongpilly, Australia.
OF RADIOACTIVE
CASTANOSPERMINE
321
3. Pan, Y. T., Ghidoni, J., and Elbein, A. D. Manuscript in preparation. 4. Saul, R., Ghidoni, J. J., Molyneux, R. J., and Elbein, A. D. (1985) Proc. Natl. Acad. Sci. USA 82, 93-97. 5. Szumilo, T., Kaushal, G. P., and Elbein, A. D. (1986) Arch. Biochem. Biophys. 247,26 l-27 1. 6. Pan, Y. T., Hori, H., Saul, R., Sanford, B. A., Molyneux, R. J., and Elbein, A. D. (1983) Biochemistry 22, 3975-3984. 7. Sasak, V. W., Ordovas, J. M., Elbein, A. D., and Berninger, R. W. (1985) Biochem. J. 232, 759-766. 8. Palamarczyk, G., and Elbein, A. D. (1985) Biothem. J. 227.795-804. 9. Merkle, R., Elbein, A. D., and Heifetz, A. (1985) J. Biol. Chem. 260, 1083-1089. 10. Evans, E. A. (1974) Tritium and Its Compounds, pp. 294-305, Wiley, New York.
BIOCHEMISTRY
Preparation
163,3 16-32 1 (I 987)
and Characterization ROY W. KEENAN,~.
of Radioactive T. PAN,ANDALAND.
Castanospermine’ ELBEIN
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284 Received October 2 1, 1986 A procedure for the preparation of tritiated castanospennine is described. The tritiated alkaloid was shown to be chromatographically identical to the native material and exhibited the same inhibitory properties. Radiolabeled castanospermine tightly bound to purified intestinal sucrase. Following gel chromatography, each mole of enzyme was shown to have bound 1 mol of the radioactive alkaloid. Cultured MDCK cells were also shown to take up the labeled castanospermine. This compound should be a useful tool in the investigation of enzymes that are responsible for the processing of glycoprotein oligosaccharides. o 1987 Academic R.ZSS, IIIC. KEY WORDS: castanospermine; tritium labeling; amyloglucosidase; processing enzymes.
Castanospermine [( lS,&S,7R,SR,8aR)1,6,7,&tetrahydroxyindolizidine] is an indolizidine alkaloid that was isolated from the seeds of the Australian legume, Castanospermum australe (1). Animals that eat these seeds develop severe gastrointestinal symptoms that may ultimately lead to death (2). These symptoms are probably due to the fact that the alkaloid is a potent inhibitor of intestinal sucrase and maltase, as well as of other cu-glucosidases (3). In addition, when injected into rats this alkaloid strongly inhibits cY-glucosidase activity in a variety of tissues, notably the liver. After daily injections of the alkaloid for 3 days, there is an almost complete absence of glycogen granules from the cytoplasm of hepatocytes, and instead the glycogen accumulates in the lysosomes (4). Thus, in these animals, castanospermine appears to cause symptoms like those of human Pompe’s disease. In addition, castanospermine inhibits the processing of the oligosaccharide chains of the N-linked glycoproteins by virtue of the fact that it inhibits glucosidase I (and also glucosidase II) (5). As a result, cells are not able to produce ’ This investigation was supported by NIH Grants 17897-l 1 to Roy W. Keenan and AM 2 1800 and GM 3 1355 to Alan D. Elbein. 0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
the typical complex chains, and their glycoproteins instead have GlcsMan7-9(GlcNAc)2 structures* (6-9). Since the alkaloid has such interesting and potentially important effects on a variety of systems, it would be of considerable value to have radioactive castanospermine in order to be able to trace its location in cells and in animals, as well as its possible metabolism. This manuscript describes the preparation of 3H-labeled castanospermine and some of the effects of this labeled alkaloid on biological systems. MATERIALS
AND METHODS
Materials. Tritiated water (1 Ci/g) was obtained from DuPont/New England Nuclear Co. and deuterium oxide (99.96% atom excess) from Aldrich Chemical Co. Castanospermine was isolated and purified from the seeds of C. australe (2). Sucrase was isolated from rat intestine and purified to homogeneity (3). All solvents and chemicals either were the best grade available or were specially purified for HPLC. Methods. Mass spectral analysis was performed by Dr. Susan Weintraub, Depart2 Abbreviations used: GlcNAc, N-acetylglucosamine; SDS, sodium dodecyl sulfate. 316
CHROMATOGRAPHIC
PREPARATION
OF RADIOACTIVE
CASTANOSPERMINE
317
ment of Pathology, on a Hewlett-Packard lute acetic acid and applied to a 0.5 X 7-cm Model 5982 mass spectrometer in combinacolumn of Dowex 50-8X NH:. The resin tion with a Hewlett-Packard Model 5933 was washed with 7 ml of water, followed by data system. Samples were introduced by elution with 50 ml of a 0 to 0.1 M NH40H means of a direct insertion probe and were gradient. Approximately 50% of the applied analyzed by electron impact at 70 meV with activity eluted in one peak as shown in Fig. 1. an ion source temperature of 180°C. VolatilApproximately equal portions of the reization of the castanospermine occurred at maining activity were eluted in the water approximately 84°C. wash or remained bound to the column. The The ‘H NMR spectra were run on a Gen- overall yield of radioactive castanospermine eral Electric QE-300 NMR spectrophotomewas estimated to be 50% based on its inhibiter by Dr. Robert Williams and Tony Her- tion of cu-glucosidase activity, and the spenandez, Chemistry Department, University cific activity was estimated to be 25.8 mCi/ of Texas (San Antonio, TX). mmol. Samples of the radioactive compound Castanospermine was analyzed by highwere incubated for 48 h at 50°C in either pressure liquid chromatography on a Waters 0.0 1 N HCl or 0.0 1 N NaOH. HPLC analysis radial compression reversed-phase column of the products of these incubations revealed (ODS 18, 10 pm). The chromatography sol- that there was no exchange of tritium with vent was 0.0 1 M sodium octanesulfonate (pH the solvent in either case, showing that the 3.8):acetonitrile (10: 1, v/v) which was run at label is sufficiently stable to use in biological 1 ml/min. Purified radioactive castanosperexperiments. mine eluted as a single peak of radioactivity RESULTS AND DISCUSSION after approximately 10 min. Radioactivity The Purification and Characterization of was determined with a flowthrough detector Radioactive Castanospermine (Flo-One-B Model 1C radioactive detector) in most chromatographic runs. A small quantity of tritiated castanosperPreparation of [3H]castanospermine. Cas- mine, prepared by exchange in tritiated tanospermine was labeled by exchange with tritiated water in the presence of Adams catalyst (10). Platinum oxide was activated by reacting with ethanolic NaBH4. An aliquot of the suspended catalyst containing approximately 1.8 mg was added to a 0.5-ml conical vial and most of the solvent was removed under a stream of nitrogen. Tritiated water (75 ~1, 75 mCi) and 6.1 mg of castanospermine were added and the Teflon-capped vial was placed in a 125 “C heating block for 22 h. Following incubation, the cooled sample was transferred to an Eppendorf centrifuge tube together with several washings. The catalyst FRACTION NUMBER was sedimented by centrifugation, and the FIG. 1. Isolation of [3H]castanospermine by ion-exsupernatant was removed. The catalyst was change chromatography. Tritiated castanospermine washed several additional times and the su- prepared as described under Materials and Methods was applied to a 7 X 70-mm column of Dowex 50 NH; and pernatants were combined. The combined extracts were acidified with acetic acid, and eluted with approximately 10 ml of water. A linear gradient of NH,OH (25 ml water in the mixing vessel and tritiated water was largely removed by lyo- 0.1 M NH,OH in the reservoir) was begun at tube 1. philizing the sample in a microlyophilization Fractions (0.7 I ml) were collected and 15-4 aliquots apparatus. The residue was dissolved in di- were assayed for radioactivity.
318
KEENAN,
PAN, AND ELBEIN
water as described under Materials and Methods, was mixed with carrier castanospermine and subjected to HPLC. Aliquots of the chromatographic eluate were analyzed for both radioactivity and ability to inhibit amyloglucosidase. Figure 2 shows that there was good correlation between the elution profile of the radioactivity and that of the inhibitory activity. In order to further characterize the product, castanospermine was carried through the same procedure on a threefold larger scale in the presence of 100% deuterium oxide. The mass spectrum of the deuterated product revealed a mixture of different products containing from one to seven deuterium atoms. The major peaks showed two to four deuterium atoms incorporated, with the largest peak being that with three protons replaced. A comparison of the NMR spectra of the deuterated sample and the starting material revealed the presence of several peaks in the deuterated samples that were not in the starting material. These were removed when the samples were purified by ion-exchange chromatography. A comparison of the NMR spectra of the starting material and of the
FIG. 2. HPLC oftritiated castanospermine. An aliquot of tritium-labeled castanospermine was mixed with 2.4 nmol of nonlabeled castanospermine and subjected to reversed-phase HPLC. Fractions (0.3 ml) were collected and analyzed for radioactivity and for inhibitory activity against cy-glucosidase. The chromatography conditions and assays were as described under Materials and Methods.
1~~~~1’~“1’~~‘(~“~1~~‘~1~“~1 4.5 4.0 3.5
3.0
2.5
2.0
1.5
PPM
B
FIG. 3. NMR spectra of castanospermine. (A) Castanospermine; (B) castanospermine incubated with deuterium oxide under conditions identical to those used to incorporate tritium. Both spectra were taken in deuterium oxide. Details of the procedure and NMR assayare given under Materials and Methods.
purified deuterated product revealed that a number of protons had been replaced. Using the assignments provided by Hohenschultz et al. (1) it was determined that the protons on the carbons bearing the hydroxyl groups did not undergo exchange. Although precise assignment could not be made in all cases, there is no doubt that most of the exchange took place with protons (Y to the tertiary amine group (carbons 3 and 5, and the methine carbon at the ring juncture; see Fig. 3). Some exchange also took place at carbon 2, but not to the extent seen with the carbons (Yto the nitrogen. It seems likely that the purified radiolabeled castanospermine is the same stereoisomer as the starting material because the compound appeared to be chromatographitally homogeneous on both ion-exchange chromatography and HPLC. A mixture of epimers would probably show different chromatographic behavior. Also, changes in the stereochemistry of the molecule should produce changes in its activity as an inhibitor; however, these were not observed. A more compelling piece of evidence against the formation of an epimeric mixture is that the NMR spectrum (Fig. 3) provides evidence
CHROMATOGRAPHIC
PREPARATION
that no deuterium exchange on carbons 1,6, 7, and 8 occurred when the reaction was carried out under the same conditions in deuterated water. Mechanisms of epimerization involving a carbonium ion or olefin would be expected to result in deuterium incorporation into these positions.
Fate of Injected Radioactive Castanospermine Tritiated castanospermine was injected into the tail vein of a 200-g female rat (2.65 nmol/g). After 24 h, the levels of radioactivity in tissue and those in the feces and urine were determined. Approximately 80% of the injected dose was recovered in the urine and cage washings. Liver, brain, pancreas, heart, lung, kidney, spleen, adrenals, ovaries, and intestine were assayed for radioactivity. Although there were some differences in the relative amounts of radioactivity in these tissues, none of the tissues contained over a fraction of a percent of the injected radioactivity. The results obtained in this experiment were typical of those in two other experiments in which rats were injected with labeled castanospermine. All the radioactivity in the urine was shown to cochromatograph with authentic samples of castanospermine. Thus, it is clear that castanospermine is absorbed very poorly by tissues in the rat, is not metabolized to any significant extent, and is rapidly excreted in the urine. In earlier investigations in which this alkaloid was shown to have an effect on liver glycosidases, much larger doses were administered over a period of several days (4).
Biological Activity of the Labeled Castanospermine The labeled castanospermine was compared to authentic castanospermine in terms of its ability to inhibit purified rat intestinal sucrase. Figure 4 shows inhibition curves comparing the effects of increasing amounts of the two castanospermine preparations against the sucrase. It can be seen that both preparations were potent inhibitors of this
OF RADIOACTIVE
319
CASTANOSPERMINE
enzyme and the inhibition curves were almost identical. Thus, in both cases, 50% inhibition occurred at about 25 to 30 rig/ml. Since one of the advantages of having labeled castanospermine would be to localize a-glucosidases within cells or tissues, we examined the binding of [3H]castanospermine to purified intestinal sucrase to determine whether the radioactivity would remain with the enzyme and how much could be bound. Various amounts of sucrase were mixed with 100 pg of labeled castanospermine and the mixture was applied to a column of Sephadex G-200. Fractions eluting from the column were assayed for radioactivity and for enzyme activity. Figure 5 shows that a symmetrical peak of radioactivity emerged from the column in the same fractions as the enzymatic activity. The height of the radioactive peak (i.e., the amount of radioactivity bound) increased in direct proportion to the amount of sucrase used. In addition, a relatively large amount of radioactivity was retained on the column and emerged in fractions 130 and beyond. These are the fractions where low-molecular-weight material elutes, and this radioactivity represents un-
-. 0
I 50 INHIBITOR
I I 100 150 CONCENTRATION
1 200
I
(nghnl)
FIG. 4. Inhibition of sucrose activity by [‘Hlcastanospermine. Purified sucrase was assayed for its ability to cleave sucrose in the presence of various amounts of authentic castanospermine or [3H]castanospermine. Reactions were initiated by the addition of sucrose, and following an incubation of 10 mitt, the amount of reducing sugar released was measured at 520 mM by Nelson’s assay.
320
KEENAN,
PAN, AND ELBEIN
bound castanospermine. A calculation of the extent of binding using the data from Fig. 5 revealed that exactly 1 mol of castanospermine was bound per mole of sucrase. An additional experiment identical to the one shown in Fig. 5 was performed, except that the enzyme was incubated with labeled castanospermine in the presence of a 43-fold excess of unlabeled castanospermine. The result of this experiment (not shown) was that the amount of enzyme-bound radioactivity was decreased to the calculated level. This is further evidence that the radioactive castanospermine has the same biological properties as the unlabeled material. We also examined the binding, or uptake, of castanospermine by MDCK cells growing on plastic dishes. In these experiments confluent monolayers were incubated with various amounts of [3H]castanospermine for 30 min. The monolayers were then washed four or five times with phosphate-buffered saline to remove the free castanospermine. The cells were released from the plates by adding 1% SDS, and the cell suspensions were counted to determine the amount of radioactivity. Figure 6 shows that [3H]castanospermine was taken up by the cells in a dose-de-
SUCRASE 4
NUMBER
FIG. 5. Binding of [3H]castanospermine to purified sucrase. Various amounts of homogeneous enzyme were mixed with 100 fig of [‘Hlcastanospermine in 0.01 M sodium phosphate buffer, pH 7.5, in a final volume of 0.6 ml as indicated in the figure. The mixture was applied to a 1.5 X lOO-cm column of Sephadex G-200. Fractions eluting from the column were assayed for radioactivity and for sucrase activity.
I
I
I
120 160 80 OF CASTANOSPERMINE
I
200 (pglml)
6. Binding of [‘Hlcastanospermine to MDCK cells. Confluent monolayers of MDCK cells were incubated with various amounts of [3H]castanospermine for 30 min in modified Eagle’s medium containing 2% fetal calf serum as indicated in the figure. The monolayers were washed thoroughly with phosphate-buffered saline and the cells were released from the plates by adding I % SDS. The cell suspensions were counted to determine the amount of ‘H bound. FIG.
pendent manner and that the radioactivity does appear to enter the cells. However, the uptake of castanospermine by the MDCK cells may not be carrier mediated, since it was not possible to block this uptake by adding unlabeled castanospermine or swainsonine; or, it is possible that the amount of unlabeled castanospermine added was not enough to dilute the label. At any rate, the fact that castanospermine is taken up by these cells should make it possible to localize the alkaloid in various cell fractions. Castanospermine is a toxic plant alkaloid that has potent effects on various biological systems, including the inhibition of processing of N-linked oligosaccharides. In order to understand how this alkaloid affects cells in culture or animals, it would be valuable to have radioactive castanospermine. This would allow one to localize castanospermine within the cell or to determine its tissue distribution and also to determine whether this compound is metabolized in animals. Since castanospermine is found only in the seeds of the Australian tree C. austrule it does not appear feasible to label this alkaloid in vivo. Also, since nothing is known about the biosynthetic pathway of this compound, it is not possible at this point to isolate or label inter-
1 14
FRACTION
I
40 AMOUNT
CHROMATOGRAPHIC
PREPARATION
mediates. Therefore, we have developed a method to label the alkaloid with tritium by an exchange method. Since the label is stable, the compound can be used for a variety of biological studies. Some biological studies with the [3H]castanospermine are demonstrated in this report. REFERENCES 1. Hohenschultz, L. D., Bell, E. A., Jewess, P. P., Leworthy, D. P., Pryce. R. J., Arnold, E., and Clardy, J. (198 1) Phyfochemistry 20, 8 1 l-8 14. 2. Nash, R. J., Evans, S. V., Fellows, L. E., and Bell, E. A. (1985) in Plant Toxicology (Seawright, A. A., Hegarty, M. P., James, L. F., and Keeler, R. F., Ed%), pp. 309-3 14, Queensland Poisonous Plant Committee, Yeerongpilly, Australia.
OF RADIOACTIVE
CASTANOSPERMINE
321
3. Pan, Y. T., Ghidoni, J., and Elbein, A. D. Manuscript in preparation. 4. Saul, R., Ghidoni, J. J., Molyneux, R. J., and Elbein, A. D. (1985) Proc. Natl. Acad. Sci. USA 82, 93-97. 5. Szumilo, T., Kaushal, G. P., and Elbein, A. D. (1986) Arch. Biochem. Biophys. 247,26 l-27 1. 6. Pan, Y. T., Hori, H., Saul, R., Sanford, B. A., Molyneux, R. J., and Elbein, A. D. (1983) Biochemistry 22, 3975-3984. 7. Sasak, V. W., Ordovas, J. M., Elbein, A. D., and Berninger, R. W. (1985) Biochem. J. 232, 759-766. 8. Palamarczyk, G., and Elbein, A. D. (1985) Biothem. J. 227.795-804. 9. Merkle, R., Elbein, A. D., and Heifetz, A. (1985) J. Biol. Chem. 260, 1083-1089. 10. Evans, E. A. (1974) Tritium and Its Compounds, pp. 294-305, Wiley, New York.