Abstract
Cytidine-5′-monophospho-N-acetylneuraminic acid:β-galactoside α2-6sialyltransferase was purified from bovine colostrum by two sequential affinity chromatography steps on CDP-ethanolamine-Sepharose and CDP-ethanolamine-(N-caproylamino-)-Sepharose, respectively. While the conditions for elution were those of Paulsonet al. [J Biol Chem (1977) 252:3256–62], the ligand of the second affinity column was coupled to Sepharose by using 6-aminocaproic acid as linker. The ease of this procedure allows rapid synthesis of bulk quantities of ligand.
Highly purified preparations of sialyltransferase were obtained which moved on gradient gel electrophoresis as a single band of 76 kDa and on dodecylsulphate electrophoresis as a single band of 54 kDa. The product of the reaction between lactose and CMP-N-acetylneuraminic acid catalyzed by the purified sialyltransferase was identified by high-resolution 500 MHz1H-NMR spectroscopy as Neu5Acα2-6Galβ1-4Glc.
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References
Corfield AP, Schauer R (1982) in Sialic Acids, Cell Biology Monographs, Vol 10, ed. Schauer R, Springer-Verlag, New York, p 205–24.
Sadler JE, Beyer TA, Oppenheimer CL, Paulson JC, Prieels JP, Rearick JI, Hill RL (1982) in Methods in Enzymology, Vol 83, ed. Ginsburg V, Academic Press, New York, p 458–514.
Barker R, Olsen KW, Shaper JH, Hill RL (1972) J Biol Chem 247:7135–47.
Berger EG, Weiser MM, Isselbacher KJ (1976) Experientia 32:690–91.
Gerber AC, Kozdrowski I, Wyss SR, Berger EG (1979) Eur J Biochem 93:453–60.
Paulson JC, Beranek WE, Hill RL (1977) J Biol Chem 252:2356–62.
Hesford FJ, Berger EG, van Halbeek H (1983) In Proc 7th Int Symp Glycoconiugates, eds. Chester MA, Heinegård D, Lundblad A, Svensson S, Secretariat, Lund, p 742–43.
Schmid K, Polis A, Hunziker K, Fricke R, Yayoshi M (1967) Biochem J 104:361–68.
Miron T, Wilchek M (1982) Anal Biochem 126:433–35.
Vliegenthart JFG, Dorland L, van Halbeek H, Haverkamp J (1982) in Sialic Acids, Cell Biology Monographs, Vol 10, ed. Schauer R, Springer-Verlag, New York, p 127–72.
Vliegenthart JFG, Dorland L, van Halbeek H (1983) Adv Carbohydr Chem Biochem 41:209–374.
Laemmli UK (1970) Nature 227:680–85.
Maniatis T, Fritsch EF, Sambrock J (1982) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, USA.
Schaffner W, Weissmann C (1973) Anal Biochem 56:502–14.
Kamerling JP, Dorland L, van Halbeek H, Vliegenthart JFG, Messer M, Schauer R (1982) Carbohydr Res 100:331–40.
Buerk RR, Eschenbruch M, Leuthard P, Steck G (1983) in Methods in Enzymology, Vol 91, eds. Hirs CHW, Timasheff SN, Academic Press, New York, p 247–54.
Miyagi T, Tsuiki S (1982) Eur J Biochem 126:253–61.
Weinstein J, de Souza-e-Silva U, Paulson JC (1982) J Biol Chem 257:13835–44.
van Halbeek H, Vliegenthart JFG, Winterwerp H, Blanken WM, van den Eijnden DH (1983) Biochem Biophys Res Commun 110:124–31.
van den Eijnden DH, Joziasse DH, Dorland L, van Halbeek H, Vliegenthart JFG, Schmid K (1980) Biochem Biophys Res Commun 92:839–45.
Paulson JC, Weinstein J, Dorland L, van Halbeek H, Vliegenthart JFG (1982) J Biol Chem 257:12734–38.
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Hesford, F.J., Berger, E.G. & Van Halbeek, H. Purification of bovine colostrumβ-galactosideα(2–6)sialyltransferase to near homogeneity by affinity chromatography. Glycoconjugate J 1, 141–153 (1984). https://doi.org/10.1007/BF01213727
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DOI: https://doi.org/10.1007/BF01213727