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A Scintillation Proximity Assay for UDP-GalNAc:Polypeptide,N-Acetylgalactosaminyltransferase
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
239, 20–24 (1996)
0285
A Scintillation Proximity Assay for UDP-GalNAc:Polypeptide, N-Acetylgalactosaminyltransferase ˚ . P. Elhammer1 C. A. Baker, R. A. Poorman, F. J. Ke´zdy, D. J. Staples, C. W. Smith, and A Pharmacia & Upjohn, Inc., 301 Henrietta Street, Kalamazoo, Michigan 49001
Received January 3, 1996
A rapid and simple method for quantitating the reaction product of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) by scintillation proximity assay (SPA) was developed. The assay quantitates the radioactivity incorporated from 3 H-labeled UDP-GalNAc into a biotin-labeled acceptor peptide, as measured after adsorption of the acceptor peptide to avidin-coated SPA beads. The acceptor peptide, PPASTSAPG (Elhammer et al. (1993) J. Biol. Chem. 268, 10029–10038) was conjugated to biotin using a di-b-alanine spacer arm. The conjugated peptide reacted readily with the enzyme and it had an apparent Km comparable to that of the parent peptide. Using a reaction mixture consisting of 4 mg of SPA beads, 17 mM acceptor, 0.5 mM nucleotide sugar, and 7.5 U/ ml enzyme, the time dependence of product formation obeyed Michaelis–Menten-type kinetics throughout the full course of the reaction—until exhaustion of the donor substrate—and the beginning portion of the reaction was sufficiently linear for calculating accurate initial rates. Analysis of the time dependency yielded an apparent Km of 0.38 { 0.12 mM for UDP-GalNAc. The assay is conveniently carried out in 96-well microtiter plates; it is ideally suited for assaying large numbers of samples and for screening large collections of chemicals for competitive inhibitors. q 1996 Academic Press, Inc.
Mucin-type O-glycosidically linked oligosaccharides have been identified in a wide variety of proteins (1, 2); they appear to be essential for a wide variety of biological functions (3, 4). The initial reaction in the biosynthesis of O-linked oligosaccharides is the transfer of N-acetylgalactosamine from the nucleotide sugar UDP-N-acetylgalactosamine to a serine or threonine residue on the ac-
ceptor polypeptide. This reaction is catalyzed by the enzyme UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase2; EC 2.4.1.41) (2). Assays for GalNAc-transferase activity typically involve incubation of the activity-containing sample with radioactively labeled UDP-GalNAc and an intact acceptor protein such as basic myelin protein, a fragment of a deglycosylated protein such as one of the various apomucins, or a synthetic peptide (5–9). The specificity of the enzyme toward the amino acid sequence of the acceptor has recently been delineated to the point where efficient acceptor peptides can be designed (8–11). Following transfer of the radioactive sugar to the polypeptide acceptor, the product is isolated and the amount of enzymatic transfer is quantitated by measuring the radioactivity incorporated into the acceptor. Thus, assays for GalNAc-transferase activity are, in principle, relatively simple, but the quantitative isolation of the glycosylated reaction product is not without its experimental problems. For peptide acceptors, separation methods employed to date include ion-exchange, size exclusion, or reversed-phase chromatography and for protein acceptors, various precipitation procedures are used, most often with trichloracetic acid. With the former, a number of chromatography columns must be prepared, equilibrated, and developed for each experiment; with the latter, extensive and precise washing procedures must be carried out in order to reduce background radioactivity. Hence, typical GalNAc-transferase assays are always laborintensive and time-consuming and any large-scale screening for GalNAc-transferase activity would greatly benefit from the development of a technically simple assay. With an experimental simplification in mind, we de2
1
To whom correspondence should be addressed. Fax: (616) 8332500.
Abbreviations used: GalNAc-transferase, UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase; SPA, scintillation proximity assay; BOC, t-butyloxycarbonyl.
20
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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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SCINTILLATION PROXIMITY ASSAY FOR GalNAc-TRANSFERASE
21
scribe in this report a scintillation proximity assay (SPA) for the quantitation of the reaction products in GalNAc-transferase assays. The SPA technology (12) has already been applied successfully to several different types of assays (13–15). For the GalNAc-transferase assay, we chose a biotinylated acceptor peptide, streptavidin-coated SPA beads, and quantitation of incorporated radioactivity on a microplate scintillation counter. The assay described in this report is ideally suited for experiments involving large numbers of samples, such as screens for compounds affecting the activity of the enzyme. MATERIALS AND METHODS
Materials. UDP-N-[1-3H]acetylgalactosamine (8.3 Ci/mmol) and streptavidin-coated SPA beads were purchased from Amersham Corp. UDP-N-acetylgalactosamine was from Sigma. The 1 cc Bond Elut C18 columns were from Varian. White Microfluor microtiter plates with round-bottom wells were purchased from Dynatech Laboratories. Recombinant, soluble GalNActransferase was prepared as described by Homa et al. (16). All other reagents were from standard sources. Preparation of acceptor peptides. Solid-phase peptide synthesis was performed on 0.5 mmol scale, utilizing OCH2 Pam resin (Applied Biosystems Inc., Foster City, CA) and an Applied Biosystems Inc. 430A Peptide Synthesizer. b-Alanine was obtained from Advanced Chemtech (Louisville, KY); the other amino acids were from Applied Biosystems Inc. The t-butyloxycarbonyl (BOC) group was used as the N-amino-protecting group during synthesis; the alcohol functions of Ser and Thr were protected by a benzyl group. Each residue was coupled twice and then capped with acetic anhydride before the next cycle of synthesis. Quantitative ninhydrin tests were performed at each cycle of the synthesis. After removing the N-terminal BOC group in the usual fashion, biotin was attached by treating the peptide– resin in DMF with N-hydroxysuccinimidobiotin (Pierce Chemical Co.). The biotin–peptide was cleaved from the resin by treatment with HF:anisole (10:1) for 1 h at 020 to 057C. The peptide resin was triturated with ether, the crude peptide was dissolved in 50% acetic acid, and the resin was removed by filtration. The filtrate was evaporated to dryness under reduced pressure and lyophilized from glacial acetic acid. The crude peptide was purified by preparative reversed-phase chromatography on a Vydac C-18 column (250 1 22.5 mm) using a water/acetonitrile gradient, with each phase containing 0.1% TFA. Homogenous fractions, as determined by analytical HPLC, were pooled and the acetonitrile was evaporated under reduced pressure; an aqueous solution of the pooled fractions was lyophilized. The purified peptide was characterized by timeof-flight mass spectroscopy which yielded solely the anticipated (M / H)/.
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FIG. 1. Structure of the biotinylated acceptor peptide.
Assays for UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity. Standard assays for measuring the rate of reaction of the biotinylated acceptor peptide were carried out as described previously (16). The 1 cc Bond Elut columns were used for isolation of the reaction product. The SPA-based assay was carried out in 96-well microtiter plates. Unless otherwise noted, the standard reaction mixture contained the following components in a final volume of 40 ml: 2 mmol imidazole, pH 7.2, 0.4 mmol MnCl2 , 19.2 pmol (135,000 cpm) UDP-[3H]GalNAc, 0.7 nmol of the acceptor peptide biotin– bAbA-PPASTSAPG, and approximately 300 mU of enzyme. Following incubation at 377C for 30 min, the reaction was quenched by the addition of 10 ml 0.5 M EDTA. One hundred microliters of a 40 mg/ml suspension of streptavidin SPA beads in PBS containing 20% glycerol was then added to each reaction mixture (well on microtiter plate) and the plate was incubated at room temperature on an orbital shaker for 2 h before measuring the incorporated radioactivity on a Packard TopCount microplate scintillation counter. RESULTS AND DISCUSSION
The primary structure of the acceptor compound used in this study is shown in Fig. 1. Conventional rate assays using reversed-phase columns for isolation of the reaction product showed that this peptide is readily glycosylated by GalNAc-transferase. Measuring initial rates with varying concentrations of this peptide, we found that the reaction obeys simple Michaelis–Menten kinetics (data not shown), and analysis of the data yielded an apparent Km Å 1.7 mM for this acceptor, quite similar to that of the unbiotinylated peptide, Km Å 6.5 mM (9). Thus, the addition of a tripeptide-linked
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BAKER ET AL.
FIG. 2. Effect of acceptor peptide concentration on the formation of reaction product. Assays were carried out in a 96-well microtiter plate and contained 2 mmol imidazole, pH 7.2, 0.4 mmol MnCl2 , 19.2 pmol UDP-[3H]-GalNAc, approximately 300 mU of enzyme and 0 to 30 mmol acceptor peptide, in a total volume of 40 ml. After incubation for 1 h at 377C, the reaction was stopped by the addition of 10 ml of 0.5 M EDTA. One hundred microliters of a 40 mg/ml suspension of streptavidin SPA beads was added and following incubation for 2 h at room temperature, the radioactivity in the wells was counted in a microtiter plate scintillation counter. Each data point represents the mean of four determinations; error bars represent standard error of the mean. The solid diagonal line to the left of the vertical line is a linear least-squares fit of the data obtained when the acceptor peptide concentration was 11 mM and lower. The horizontal solid line was obtained by calculating the mean dpm when the acceptor peptide concentration was 17 mM and higher.
biotin to the NH2-terminus of the peptide PPASTSAPG did not affect adversely the reactivity of this acceptor toward GalNAc-transferase. In fact, the slight but significant decrease in Km with respect to that of the unbiotinylated peptide indicates a binding contribution of the N-terminal extension. In other words, the binding site of the enzyme may well extend beyond the four residues postulated to participate in substrate binding. Alternatively, the two-amino-acid linker may simply negate an unfavorable influence of the free amino terminal of the unbiotinylated acceptor. An enzymatic assay is ideally suited for measuring the activity of competitive inhibitors when the substrate concentration is below Km . For this reason, but also because of the limited capacity of the streptavidincoated SPA beads that could be used in this study (115 pmol/mg), the acceptor concentrations in our assays are far below saturation. The most suitable acceptor concentration in the assays employing 4 mg SPA beads—the maximal amount useable in 96-well plates—was determined from the experiment shown in Fig. 2. At that bead concentration, the incorporation of radioactivity, as measured by SPA, depended linearly on the substrate concentration. This was as ex-
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pected for Michaelis–Menten-type kinetics with S0 p Km . A limiting value was, however, reached at a concentration of approximately 17 mM peptide, still two orders of magnitude below Km . This plateau of adsorption must therefore reflect saturation of the SPA beads. This conclusion is also borne out by the results shown in Fig. 3 where increasing amounts of SPA beads were added to assay mixtures all containing the same amount of reaction products; maximal radioactive signal was obtained using 4 mg of beads. Indeed, lowering the amount of SPA beads resulted in a linear decrease in detected radioactivity, i.e., the amount of SPA beads added is clearly exceeded by the amount of radioactive product in this reaction mixture. Nevertheless, at acceptor concentrations up to 17 mM, the assay with 4 mg SPA beads shows a typical Michaelis–Menten-type dependency on the limiting donor concentration. For example, analysis of the data shown in Fig. 4 demonstrates full conformity with Michaelis–Menten kinetics, as shown by the agreement between the experimental points and the theoretical curve calculated on the basis of the hyperbolic rate law and the best-fit parameters obtained by a nonlinear least-squares algorithm. The analysis yielded an apparent Km of 0.8 mM for UDPGalNAc. Keeping the donor concentration slightly below the capacity of the beads, we also determined the time dependency of the reaction. As shown in Fig. 5, the reaction curve is, again, typical for integrated Michaelis–Menten-type behavior, as indicated by the largely linear shape of the product vs time curve for most of the duration of the reaction. Using the integrated form of the Michaelis–Menten equation and a
FIG. 3. Effect of SPA bead concentration on the recovery of radioactivity from the reaction product. Assays conditions were as described in the legend to Fig. 2. The acceptor concentration was 17 mM and the amount of SPA bead suspension added to the assay was varied from 0 to 100 ml (4 mg). Each data point represents the mean of three determinations; error bars represent standard error of the mean. The solid line is a linear least-squares fit to the data.
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SCINTILLATION PROXIMITY ASSAY FOR GalNAc-TRANSFERASE
FIG. 4. Effect of UDP-GalNAc concentration on the formation of reaction product. Assay conditions were as described in the legend to Fig. 2; the acceptor concentration was 17 mM. The amount of UDP[3H]-GalNAc varied from 0 to 16 pmol. The solid curve is a nonlinear least-squares fit to the Michaelis–Menten equation, where Vmax Å 37.8 { 4.6 1 103 dpm/h and Km Å 0.8 { 0.2 mM.
nonlinear least-squares-fit method, we calculate Km Å 0.38 { 0.12 mM for UDP-GalNAc under our experimental conditions. The agreement of the experimental data and the theoretical curve based on the best-fit parameters of the Michaelis–Menten equation indicate that the reaction is uncomplicated by substrate inhibition or by enzyme decomposition. The slight curvature of
23
FIG. 6. Effect of enzyme concentration on the formation of reaction product. Assay conditions were as described in the legend to Fig. 2; the acceptor concentration was 17 mM. The amount of enzyme in the assays was varied from 0 to 600 mU. Each data point represents the mean of three determinations; error bars represent standard error of the mean. The solid line is a linear least-squares fit to the data.
the reaction during the first 60 min shows that the quantity of enzyme used in this assay is very probably an upper limit where linearity of the assay velocity versus enzyme concentration still obtains. A final variable investigated during validation of this assay was the quantity of enzyme used. As can be seen in Fig. 6, the addition of increasing amounts of enzyme resulted in a linear increase in product formation up to at least 600 mU; the standard assay uses approximately 300 mU of enzyme. In conclusion, the assay described in this report provides a fast, simple, and reproducible way of measuring GalNAc-transferase activity. With longer incubation times, the sensitivity of the assay could be increased by more than an order of magnitude. Because of the high specificity of the biotin–avidin interaction, the probability of artifacts arising from contaminants interfering with binding of the reaction products is rather low. Finally, large numbers of samples can easily be processed and, using the recombinant enzyme, excellent signal-to-noise ratios (typically ú15:1) are obtained. The availability of this assay should greatly facilitate screening for GalNAc-transferase-specific competitive inhibitors. REFERENCES
FIG. 5. Effect of incubation time on the formation of reaction product. Assay conditions were as described in the legend to Fig. 2; the acceptor concentration was 17 mM. The incubation time was varied from 0 to 3 h. The solid curve is a nonlinear least-squares fit to an integrated form of the Michaelis–Menten equation, where the Km for GalNAc was found to be 0.38 { 0.12 mM.
1. Sadler, J. E. (1984) in Biology of Carbohydrates (Ginsburg, V., and Robbins, P. W., Eds.) Vol. 2, pp. 199–213, Wiley, New York. 2. Schachter, H., and Brockhausen, I. (1992) in Glycoconjugates (Allen, H. J., and Kisailu, E. C., Eds.) pp. 263–232, Dekker, New York. 3. Paulson, J. C. (1989) Trends Biochem. Sci. 14, 272–275.
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4. Jentoft, N. (1990) Trends Biochem. Sci. 15, 291–294. 5. Hagopian, A., Westall, F. C., Whitehead, J. S., and Eylar, E. H. (1971) J. Biol. Chem. 246, 2519–2523. 6. Hagopian, A., and Eylar, E. H. (1968) Arch. Biochem. Biophys. 128, 422–433. 7. Young, J. D., Tsuchiya, D., Sandlin, D. E., and Holroyde, M. J. (1979) Biochemistry 18, 4444–4448. 8. Wang, Y., Agrwal, N., Eckhardt, A. E., Stevens, R. D., and Hill, R. L. (1993) J. Biol. Chem. 268, 22979–22983. ˚ . P., Poorman, R. A., Brown, E., Maggiora, L. L., 9. Elhammer, A Hoogerheide, J. G., and Ke´zdy, F. J. (1993) J. Biol. Chem. 268, 10029–10038. 10. O’Connell, B. C., Hagen, F. K., and Tabak, L. A. (1992) J. Biol. Chem. 267, 25010–25018.
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11. Hansen, J. E., Lund, O., Engelbrecht, J., Bohr, H., Nielsen, J. O., Hansen, J.-E. S., and Brunak, S. (1995) Biochem. J. 308, 801–813. 12. Bosworth, N., and Towers, P. (1989) Nature 341, 167–168. 13. Lemaitre, M., Phan, T., Downes, M. J., Williams, K. B., and Cook, N. D. (1992) Antiviral Res. 17(Suppl. 1), 48. 14. Fehrentz, J. A., Chomier, B., Bignon, E., Venaud, S., Chermann, J. C., and Nisato, D. (1992) Biochim. Biophys. Res. Commun. 188, 865–872. 15. Brown, A. M., George, S. M., Blume, A. J., Dushin, R. G., Jacobsen, S., and Sonnenberg-Reines, J. (1994) Anal. Biochem. 217, 139–147. 16. Homa, F. L., Baker, C. A., Thomsen, D. R., and Elhammer, ˚ . P. (1995) Protein Express. Purif. 6, 141–148. A
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AP: Anal Bio
239, 20–24 (1996)
0285
A Scintillation Proximity Assay for UDP-GalNAc:Polypeptide, N-Acetylgalactosaminyltransferase ˚ . P. Elhammer1 C. A. Baker, R. A. Poorman, F. J. Ke´zdy, D. J. Staples, C. W. Smith, and A Pharmacia & Upjohn, Inc., 301 Henrietta Street, Kalamazoo, Michigan 49001
Received January 3, 1996
A rapid and simple method for quantitating the reaction product of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) by scintillation proximity assay (SPA) was developed. The assay quantitates the radioactivity incorporated from 3 H-labeled UDP-GalNAc into a biotin-labeled acceptor peptide, as measured after adsorption of the acceptor peptide to avidin-coated SPA beads. The acceptor peptide, PPASTSAPG (Elhammer et al. (1993) J. Biol. Chem. 268, 10029–10038) was conjugated to biotin using a di-b-alanine spacer arm. The conjugated peptide reacted readily with the enzyme and it had an apparent Km comparable to that of the parent peptide. Using a reaction mixture consisting of 4 mg of SPA beads, 17 mM acceptor, 0.5 mM nucleotide sugar, and 7.5 U/ ml enzyme, the time dependence of product formation obeyed Michaelis–Menten-type kinetics throughout the full course of the reaction—until exhaustion of the donor substrate—and the beginning portion of the reaction was sufficiently linear for calculating accurate initial rates. Analysis of the time dependency yielded an apparent Km of 0.38 { 0.12 mM for UDP-GalNAc. The assay is conveniently carried out in 96-well microtiter plates; it is ideally suited for assaying large numbers of samples and for screening large collections of chemicals for competitive inhibitors. q 1996 Academic Press, Inc.
Mucin-type O-glycosidically linked oligosaccharides have been identified in a wide variety of proteins (1, 2); they appear to be essential for a wide variety of biological functions (3, 4). The initial reaction in the biosynthesis of O-linked oligosaccharides is the transfer of N-acetylgalactosamine from the nucleotide sugar UDP-N-acetylgalactosamine to a serine or threonine residue on the ac-
ceptor polypeptide. This reaction is catalyzed by the enzyme UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase2; EC 2.4.1.41) (2). Assays for GalNAc-transferase activity typically involve incubation of the activity-containing sample with radioactively labeled UDP-GalNAc and an intact acceptor protein such as basic myelin protein, a fragment of a deglycosylated protein such as one of the various apomucins, or a synthetic peptide (5–9). The specificity of the enzyme toward the amino acid sequence of the acceptor has recently been delineated to the point where efficient acceptor peptides can be designed (8–11). Following transfer of the radioactive sugar to the polypeptide acceptor, the product is isolated and the amount of enzymatic transfer is quantitated by measuring the radioactivity incorporated into the acceptor. Thus, assays for GalNAc-transferase activity are, in principle, relatively simple, but the quantitative isolation of the glycosylated reaction product is not without its experimental problems. For peptide acceptors, separation methods employed to date include ion-exchange, size exclusion, or reversed-phase chromatography and for protein acceptors, various precipitation procedures are used, most often with trichloracetic acid. With the former, a number of chromatography columns must be prepared, equilibrated, and developed for each experiment; with the latter, extensive and precise washing procedures must be carried out in order to reduce background radioactivity. Hence, typical GalNAc-transferase assays are always laborintensive and time-consuming and any large-scale screening for GalNAc-transferase activity would greatly benefit from the development of a technically simple assay. With an experimental simplification in mind, we de2
1
To whom correspondence should be addressed. Fax: (616) 8332500.
Abbreviations used: GalNAc-transferase, UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase; SPA, scintillation proximity assay; BOC, t-butyloxycarbonyl.
20
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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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aba
AP: Anal Bio
SCINTILLATION PROXIMITY ASSAY FOR GalNAc-TRANSFERASE
21
scribe in this report a scintillation proximity assay (SPA) for the quantitation of the reaction products in GalNAc-transferase assays. The SPA technology (12) has already been applied successfully to several different types of assays (13–15). For the GalNAc-transferase assay, we chose a biotinylated acceptor peptide, streptavidin-coated SPA beads, and quantitation of incorporated radioactivity on a microplate scintillation counter. The assay described in this report is ideally suited for experiments involving large numbers of samples, such as screens for compounds affecting the activity of the enzyme. MATERIALS AND METHODS
Materials. UDP-N-[1-3H]acetylgalactosamine (8.3 Ci/mmol) and streptavidin-coated SPA beads were purchased from Amersham Corp. UDP-N-acetylgalactosamine was from Sigma. The 1 cc Bond Elut C18 columns were from Varian. White Microfluor microtiter plates with round-bottom wells were purchased from Dynatech Laboratories. Recombinant, soluble GalNActransferase was prepared as described by Homa et al. (16). All other reagents were from standard sources. Preparation of acceptor peptides. Solid-phase peptide synthesis was performed on 0.5 mmol scale, utilizing OCH2 Pam resin (Applied Biosystems Inc., Foster City, CA) and an Applied Biosystems Inc. 430A Peptide Synthesizer. b-Alanine was obtained from Advanced Chemtech (Louisville, KY); the other amino acids were from Applied Biosystems Inc. The t-butyloxycarbonyl (BOC) group was used as the N-amino-protecting group during synthesis; the alcohol functions of Ser and Thr were protected by a benzyl group. Each residue was coupled twice and then capped with acetic anhydride before the next cycle of synthesis. Quantitative ninhydrin tests were performed at each cycle of the synthesis. After removing the N-terminal BOC group in the usual fashion, biotin was attached by treating the peptide– resin in DMF with N-hydroxysuccinimidobiotin (Pierce Chemical Co.). The biotin–peptide was cleaved from the resin by treatment with HF:anisole (10:1) for 1 h at 020 to 057C. The peptide resin was triturated with ether, the crude peptide was dissolved in 50% acetic acid, and the resin was removed by filtration. The filtrate was evaporated to dryness under reduced pressure and lyophilized from glacial acetic acid. The crude peptide was purified by preparative reversed-phase chromatography on a Vydac C-18 column (250 1 22.5 mm) using a water/acetonitrile gradient, with each phase containing 0.1% TFA. Homogenous fractions, as determined by analytical HPLC, were pooled and the acetonitrile was evaporated under reduced pressure; an aqueous solution of the pooled fractions was lyophilized. The purified peptide was characterized by timeof-flight mass spectroscopy which yielded solely the anticipated (M / H)/.
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FIG. 1. Structure of the biotinylated acceptor peptide.
Assays for UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity. Standard assays for measuring the rate of reaction of the biotinylated acceptor peptide were carried out as described previously (16). The 1 cc Bond Elut columns were used for isolation of the reaction product. The SPA-based assay was carried out in 96-well microtiter plates. Unless otherwise noted, the standard reaction mixture contained the following components in a final volume of 40 ml: 2 mmol imidazole, pH 7.2, 0.4 mmol MnCl2 , 19.2 pmol (135,000 cpm) UDP-[3H]GalNAc, 0.7 nmol of the acceptor peptide biotin– bAbA-PPASTSAPG, and approximately 300 mU of enzyme. Following incubation at 377C for 30 min, the reaction was quenched by the addition of 10 ml 0.5 M EDTA. One hundred microliters of a 40 mg/ml suspension of streptavidin SPA beads in PBS containing 20% glycerol was then added to each reaction mixture (well on microtiter plate) and the plate was incubated at room temperature on an orbital shaker for 2 h before measuring the incorporated radioactivity on a Packard TopCount microplate scintillation counter. RESULTS AND DISCUSSION
The primary structure of the acceptor compound used in this study is shown in Fig. 1. Conventional rate assays using reversed-phase columns for isolation of the reaction product showed that this peptide is readily glycosylated by GalNAc-transferase. Measuring initial rates with varying concentrations of this peptide, we found that the reaction obeys simple Michaelis–Menten kinetics (data not shown), and analysis of the data yielded an apparent Km Å 1.7 mM for this acceptor, quite similar to that of the unbiotinylated peptide, Km Å 6.5 mM (9). Thus, the addition of a tripeptide-linked
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FIG. 2. Effect of acceptor peptide concentration on the formation of reaction product. Assays were carried out in a 96-well microtiter plate and contained 2 mmol imidazole, pH 7.2, 0.4 mmol MnCl2 , 19.2 pmol UDP-[3H]-GalNAc, approximately 300 mU of enzyme and 0 to 30 mmol acceptor peptide, in a total volume of 40 ml. After incubation for 1 h at 377C, the reaction was stopped by the addition of 10 ml of 0.5 M EDTA. One hundred microliters of a 40 mg/ml suspension of streptavidin SPA beads was added and following incubation for 2 h at room temperature, the radioactivity in the wells was counted in a microtiter plate scintillation counter. Each data point represents the mean of four determinations; error bars represent standard error of the mean. The solid diagonal line to the left of the vertical line is a linear least-squares fit of the data obtained when the acceptor peptide concentration was 11 mM and lower. The horizontal solid line was obtained by calculating the mean dpm when the acceptor peptide concentration was 17 mM and higher.
biotin to the NH2-terminus of the peptide PPASTSAPG did not affect adversely the reactivity of this acceptor toward GalNAc-transferase. In fact, the slight but significant decrease in Km with respect to that of the unbiotinylated peptide indicates a binding contribution of the N-terminal extension. In other words, the binding site of the enzyme may well extend beyond the four residues postulated to participate in substrate binding. Alternatively, the two-amino-acid linker may simply negate an unfavorable influence of the free amino terminal of the unbiotinylated acceptor. An enzymatic assay is ideally suited for measuring the activity of competitive inhibitors when the substrate concentration is below Km . For this reason, but also because of the limited capacity of the streptavidincoated SPA beads that could be used in this study (115 pmol/mg), the acceptor concentrations in our assays are far below saturation. The most suitable acceptor concentration in the assays employing 4 mg SPA beads—the maximal amount useable in 96-well plates—was determined from the experiment shown in Fig. 2. At that bead concentration, the incorporation of radioactivity, as measured by SPA, depended linearly on the substrate concentration. This was as ex-
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pected for Michaelis–Menten-type kinetics with S0 p Km . A limiting value was, however, reached at a concentration of approximately 17 mM peptide, still two orders of magnitude below Km . This plateau of adsorption must therefore reflect saturation of the SPA beads. This conclusion is also borne out by the results shown in Fig. 3 where increasing amounts of SPA beads were added to assay mixtures all containing the same amount of reaction products; maximal radioactive signal was obtained using 4 mg of beads. Indeed, lowering the amount of SPA beads resulted in a linear decrease in detected radioactivity, i.e., the amount of SPA beads added is clearly exceeded by the amount of radioactive product in this reaction mixture. Nevertheless, at acceptor concentrations up to 17 mM, the assay with 4 mg SPA beads shows a typical Michaelis–Menten-type dependency on the limiting donor concentration. For example, analysis of the data shown in Fig. 4 demonstrates full conformity with Michaelis–Menten kinetics, as shown by the agreement between the experimental points and the theoretical curve calculated on the basis of the hyperbolic rate law and the best-fit parameters obtained by a nonlinear least-squares algorithm. The analysis yielded an apparent Km of 0.8 mM for UDPGalNAc. Keeping the donor concentration slightly below the capacity of the beads, we also determined the time dependency of the reaction. As shown in Fig. 5, the reaction curve is, again, typical for integrated Michaelis–Menten-type behavior, as indicated by the largely linear shape of the product vs time curve for most of the duration of the reaction. Using the integrated form of the Michaelis–Menten equation and a
FIG. 3. Effect of SPA bead concentration on the recovery of radioactivity from the reaction product. Assays conditions were as described in the legend to Fig. 2. The acceptor concentration was 17 mM and the amount of SPA bead suspension added to the assay was varied from 0 to 100 ml (4 mg). Each data point represents the mean of three determinations; error bars represent standard error of the mean. The solid line is a linear least-squares fit to the data.
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SCINTILLATION PROXIMITY ASSAY FOR GalNAc-TRANSFERASE
FIG. 4. Effect of UDP-GalNAc concentration on the formation of reaction product. Assay conditions were as described in the legend to Fig. 2; the acceptor concentration was 17 mM. The amount of UDP[3H]-GalNAc varied from 0 to 16 pmol. The solid curve is a nonlinear least-squares fit to the Michaelis–Menten equation, where Vmax Å 37.8 { 4.6 1 103 dpm/h and Km Å 0.8 { 0.2 mM.
nonlinear least-squares-fit method, we calculate Km Å 0.38 { 0.12 mM for UDP-GalNAc under our experimental conditions. The agreement of the experimental data and the theoretical curve based on the best-fit parameters of the Michaelis–Menten equation indicate that the reaction is uncomplicated by substrate inhibition or by enzyme decomposition. The slight curvature of
23
FIG. 6. Effect of enzyme concentration on the formation of reaction product. Assay conditions were as described in the legend to Fig. 2; the acceptor concentration was 17 mM. The amount of enzyme in the assays was varied from 0 to 600 mU. Each data point represents the mean of three determinations; error bars represent standard error of the mean. The solid line is a linear least-squares fit to the data.
the reaction during the first 60 min shows that the quantity of enzyme used in this assay is very probably an upper limit where linearity of the assay velocity versus enzyme concentration still obtains. A final variable investigated during validation of this assay was the quantity of enzyme used. As can be seen in Fig. 6, the addition of increasing amounts of enzyme resulted in a linear increase in product formation up to at least 600 mU; the standard assay uses approximately 300 mU of enzyme. In conclusion, the assay described in this report provides a fast, simple, and reproducible way of measuring GalNAc-transferase activity. With longer incubation times, the sensitivity of the assay could be increased by more than an order of magnitude. Because of the high specificity of the biotin–avidin interaction, the probability of artifacts arising from contaminants interfering with binding of the reaction products is rather low. Finally, large numbers of samples can easily be processed and, using the recombinant enzyme, excellent signal-to-noise ratios (typically ú15:1) are obtained. The availability of this assay should greatly facilitate screening for GalNAc-transferase-specific competitive inhibitors. REFERENCES
FIG. 5. Effect of incubation time on the formation of reaction product. Assay conditions were as described in the legend to Fig. 2; the acceptor concentration was 17 mM. The incubation time was varied from 0 to 3 h. The solid curve is a nonlinear least-squares fit to an integrated form of the Michaelis–Menten equation, where the Km for GalNAc was found to be 0.38 { 0.12 mM.
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