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For the 3D structure of the β1,4GalT, see: Gastinel, L. N.; Cambillau, C.; Bourne, Y. EMBO J. 1999, 18, 3546.
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19
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In the course of these studies, information was also provided on the 3D structure of the α1,3GalT, substrates' binding and catalytic mechanism. See: Boix, E, Zhang, Y, Swaminathan, G. J, Brew, K, Acharya, K. R. J. Biol. Chem. 2002, 277, 28310
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In the course of these studies, information was also provided on the 3D structure of the α1,3GalT, substrates' binding and catalytic mechanism. See: Boix, E.; Zhang, Y.; Swaminathan, G. J.; Brew, K.; Acharya, K. R. J. Biol. Chem. 2002, 277, 28310.
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For a recent disclosure of a static uridine-based library targeting the N-acetyl-α-galactosaminyltransferases, see: Hang, H. C.; Yu, C.; Ten Hagen, K. G.; Tian, E.; Winans, K. A.; Tabak, L. A.; Bertozzi, C. R. Chem. Biol. 2004, 11, 337.
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33846585127
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We have previously checked that each enzyme was still active after 9 or 13 days only under these buffer conditions, and for the study with the DCL members, we have kept those conditions (buffer, pH) that are optimal for the activity of each enzyme
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We have previously checked that each enzyme was still active after 9 or 13 days only under these buffer conditions, and for the study with the DCL members, we have kept those conditions (buffer, pH) that are optimal for the activity of each enzyme.
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32
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25144520090
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Unfortunately, all attempts to identify all the members of the library by LC/MS or LC/evaporative light scattering methods failed. For monitoring a glycopeptide DCL by ESI-MS, see: Hotchkiss, T.; Kramer, H. B.; Doores, K. J.; Gamblin, D. P.; Oldham, N. J.; Davis, B. G. Chem. Commun. 2005, 4264.
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Unfortunately, all attempts to identify all the members of the library by LC/MS or LC/evaporative light scattering methods failed. For monitoring a glycopeptide DCL by ESI-MS, see: Hotchkiss, T.; Kramer, H. B.; Doores, K. J.; Gamblin, D. P.; Oldham, N. J.; Davis, B. G. Chem. Commun. 2005, 4264.
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33
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33846571504
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Although the α1,3GalT was able to induce amplifications with an unusually small concentration (1.6 μM, 10 times more of the β1,4GalT (16 μM) was needed to detect amplifications
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Although the α1,3GalT was able to induce amplifications with an unusually small concentration (1.6 μM), 10 times more of the β1,4GalT (16 μM) was needed to detect amplifications.
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34
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33846632908
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Setup of the DCLs: Recombinant bovine α1,3GalT was purchased from Calbiochem. A solution of bovine β1,4GalT (Calbiochem) was prepared by dissolving the lyophilized recombinant enzyme (0.54 mg) in 500 μL of MOPS buffer (50 mM, pH 7.4) containing MnCl2 (20 mM, Stock solutions of amines A-D (10 mM each, aldehydes 1 and 2 (hydrated form, 2 mM each, and sodium cyanoborohydride (50 mM) were prepared in distilled water. Conditions for the α1,3GalT enzyme: Solutions of amines A-D (12 μL, aldehydes 1 and 2 (12 μL, and sodium cyanoborohydride (7 μL) were introduced into an Eppendorf tube containing a solution of α1,3GalT in a Tris-acetate buffer (25 mM, pH 7.9, MgCl 2 (10 mM, and 50% v/v of glycerol (200 μL, A control experiment was performed in a Tris-acetate buffer (25 mM, pH 7.9, MgCl2 (10 mM, and 50% v/v of glycerol without the enzyme. The resulting mixtures (295 μL) w
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2 (20 mM) without the enzyme. The resulting mixtures (100 μL) were equilibrated at ambient temperature for 9 days. Aliquots (20 μL) were removed and diluted with acetic acid (10 μL).
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