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Purification of a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase to homogeneity from rat liver

Weinstein, J ; de Souza-e-Silva, U ; Paulson, J C

The Journal of biological chemistry, 1982-11, Vol.257 (22), p.13835-13844 [Periódico revisado por pares]

United States: Elsevier Inc

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  • Título:
    Purification of a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase to homogeneity from rat liver
  • Autor: Weinstein, J ; de Souza-e-Silva, U ; Paulson, J C
  • Assuntos: Animals ; Antigen-Antibody Complex ; Carbohydrate Conformation ; Chromatography, Affinity ; Immune Sera ; Kinetics ; Liver - enzymology ; Molecular Weight ; Rats ; Sialyltransferases - isolation & purification ; Sialyltransferases - metabolism ; Substrate Specificity ; Transferases - isolation & purification
  • É parte de: The Journal of biological chemistry, 1982-11, Vol.257 (22), p.13835-13844
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
  • Descrição: A Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferse and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase have been purified 23,000- and 860,000-fold to homogeneity from Triton CF-54 extracts of rat liver membranes. The two enzymes were concentrated by affinity chromatography on CDP-hexanolamine-agarose and resolved by NaCl gradient elution from the same adsorbent. Final purification of the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialytransferase, the most abundant enzyme, was achieved by specific elution from CDP-agarose with CDP. The Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase was also purified further by CDP elution from CDP-agarose, but final purification required affinity chromatography on an adsorbent prepared by coupling asialoprothrombin to cyanogen bromide-activated agarose. Asialoprothrombin contains the terminal sequence Gal beta 1 to 3GlcNAc on N-linked oligosaccharides and is the best acceptor substrate of the enzyme (Km congruent to 6 microM). The Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase was found to bind to asialoprothrombin-agarose in the presence of CDP and could be eluted with a solution containing 0.2 M lactose and no CDP. Sodium dodecyl sulfate-gel electrophoresis of the Gal beta 1 to 4GlcNAc alpha 2 to 6 and Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferases revealed a single major protein band for each enzyme with apparent molecular weights of 40,500 and 44,000, respectively. Rabbit antibodies raised to the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase inhibit its enzymatic activity greater than 99% but caused little or no inhibition of Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialytransferase. Moreover, the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase quantitatively bound to a column containing antibody adsorbed to Protein A-agarose, while the Gal beta 1 to 3(4) GlcNAc alpha 2 to 3 sialyltransferase did not bind. This demonstrated that the two sialyltransferases are antigenically unrelated and formed the basis for removal of contaminating Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase from solutions of the Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase. Enzymatic characterization of the two sialyltransferases suggests that their major biological roles are in the terminal glycosylation of N-linked oligosaccharides of glycoproteins. (Weinstein, J., de Souza-e-Silva, U., and Paulson J. C. (1982) J. Biol. Chem. 257, 13845-13853. The alpha 2 to 6 sialyltransferase efficiently forms the NeuAc alpha 2 to 6Gal beta 1 to 4GlcNAc sequence, and the alpha 2 to 3 sialyltransferase forms the NeuAc alpha 2 to 3Gal beta 1 to 3GlcNAc and NeuAc alpha 2 to 3Ga; beta 1 to 4GlcNAc sequences.
  • Editor: United States: Elsevier Inc
  • Idioma: Inglês
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