Purification and characterization of CMP-N-acetylneuraminic acid:lactosylceramide (α2-3) sialyltransferase (G(M3)-synthase) from rat brain

U. Preuss, X. Gu, T. Gu, R. K. Yu

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

CMP-N-acetylneuraminic acid:lactosylceramide (α2-3) sialyltransferase (G(M3)-synthase) was purified to homogeneity from a Triton CF-54 extract of young rat brain. The enzyme was separated by affinity chromatography on CDP- Sepharose column and resolved by linear NaCl gradient elution from the same adsorbent. Final purification of G(M3)-synthase was achieved by chromatography on a 'lactosylceramide acid'-Sepharose column and specific elution with lactosylceramide. The enzyme activity was highest at pH 6.5 and required the presence of Triton CF-54 (0.15%) and Mn2+ (10 mM) for its full activity. The product of the reaction catalyzed by the enzyme was identified as G(M3) based on its mobility on thin layer chromatographic plates using two different solvent systems. Comparison with several glycolipid substrates showed high specificity of G(M3)-synthase for lactosylceramide. The apparent K(m) value for lactosylceramide and CMP-N-acetylneuraminic acid were 80 and 210 μM, respectively. The apparent molecular mass of the enzyme determined on SDS-polyacrylamide gel electrophoresis was 76 kDa.

Original languageEnglish (US)
Pages (from-to)26273-26278
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number35
StatePublished - Jan 1 1993
Externally publishedYes

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Sialyltransferases
Cytidine Monophosphate
N-Acetylneuraminic Acid
Purification
Rats
Brain
Enzymes
Sepharose
Cytidine Diphosphate
Affinity chromatography
Glycolipids
Enzyme activity
Molecular mass
Chromatography
Electrophoresis
Affinity Chromatography
Adsorbents
Polyacrylamide Gel Electrophoresis
Acids
CDw17 antigen

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification and characterization of CMP-N-acetylneuraminic acid:lactosylceramide (α2-3) sialyltransferase (G(M3)-synthase) from rat brain. / Preuss, U.; Gu, X.; Gu, T.; Yu, R. K.

In: Journal of Biological Chemistry, Vol. 268, No. 35, 01.01.1993, p. 26273-26278.

Research output: Contribution to journalArticle

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