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Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain

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Abstract

A β1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 β1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, α-lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAcβ1-4Galβ-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl2, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.

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Abbreviations

CNS:

central nervous system

GM1:

monosialotetraosylganglioside, Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-1Cer

GM2:

monosialotriaosylganglioside, GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glcβ1-1Cer

DSS:

detergent solubilized supernatant

ECB:

embryonic chicken brain

TBS:

Tris-buffered saline

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Authors and Affiliations

  1. Department of Molecular Cancer Biology, Duke University Medical Center, 27710, Durham, NC, USA

    Sujoy Ghosh

  2. Department of Cardiology, University of Chicago, 60637, Chicago, IL, USA

    John W. Kyle

  3. Centre de Biophysique Moleculaire, CNRS, 45071, Orleans, Cedex 2, France

    Francois Daussin

  4. Department of Chemistry and Biochemistry, Biochemistry, Biophysics and Molecular Biology Program, University of Notre Dame, 46556, Notre Dame, IN, USA

    Sara Dastgheib, Zhixiong Li & Subhash Basu

Authors
  1. Sujoy Ghosh
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  2. John W. Kyle
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  3. Sara Dastgheib
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  4. Francois Daussin
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  5. Zhixiong Li
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  6. Subhash Basu
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Ghosh, S., Kyle, J.W., Dastgheib, S. et al. Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain. Glycoconjugate J 12, 838–847 (1995). https://doi.org/10.1007/BF00731246

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  • DOI: https://doi.org/10.1007/BF00731246

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