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A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

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Abstract

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C18 cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar GM3-ganglioside is simple and rapid relative to previously described methods. Recovery of GM3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (GM3 synthase; ST-1), the transfer of [14C] sialic acid from CMP-[14C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.

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Abbreviations

GM3 :

GM3-ganglioside

II3NeuAc-LacCer:

NeuAcα2-3Galβ1-4Glcβ1-1Cer

GD1a :

GD1a-ganglioside, IV3NeuAc, II3NeuAc-GgOse4Cer, NeuAcα2-3Galβ1-3GalNacβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-1Cer

GD3 :

GD3-ganglioside, II3(NeuAc)2LacCer, NeuAcα2-8NeuAcα2-3Galβ1-4Glcβ1-1Cer

GgOse4Cer:

asialo-GM1 Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer

FucGMI :

fucosyl-GMI-ganglioside, Fucα1-2Galβ1-3GalNAcβ1-4Galβ1-4 Glcβ1-1Cer

ST-1:

GM3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase

LacCer:

lactosylceramide, Galβ1-4Glcβ1-1Cer

CMP-NeuAc:

cytidine 5′-monophospho-N-acetylneuraminic acid

PC:

phosphatidylcholine

PMSF:

phenylmethylsulfonyl fluoride

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Authors and Affiliations

  1. Department of Biochemistry, Michigan State University, 48824, East Lansing, MI, USA

    Lyla J Melkerson-Watson, Kimihiro Kanemitsu & Charles C Sweeley

Authors
  1. Lyla J Melkerson-Watson
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  2. Kimihiro Kanemitsu
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  3. Charles C Sweeley
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Melkerson-Watson, L.J., Kanemitsu, K. & Sweeley, C.C. A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges. Glycoconjugate J 4, 7–16 (1987). https://doi.org/10.1007/BF01048440

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  • DOI: https://doi.org/10.1007/BF01048440

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