Purification and characterization of CMP-N-acetylneuraminic acid:Lactosylceramide (α2-3) sialyltransferase (G_M3-synthase) from rat brain

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.