PURIFICATION AND CHARACTERIZATION OF CMP-N-ACETYLNEURAMINIC ACID-LACTOSYLCERAMIDE (ALPHA-2-3) SIALYLTRANSFERASE (G(M3)-SYNTHASE) FROM RAT-BRAIN

CMP-N-acetylneuraminic acid:lactosylceramide (alpha2-3) sialyltransfer ase (G(M3)-synthase) was purified to homogeneity from a Triton CF-54 e xtract of young rat brain. The enzyme was separated by affinity chroma tography 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''-Sepharos e column and specific elution with lactosylceramide. The enzyme activi ty 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 reacti on catalyzed by the enzyme was identified as G(M3) based on its mobili ty on thin layer chromatographic plates using two different solvent sy stems. Comparison with several glycolipid substrates showed high speci ficity of G(M3)-synthase for lactosylceramide. The apparent K(m) value for lactosylceramide and CMP-N-acetylneuraminic acid were 80 and 210 muM, respectively. The apparent molecular mass of the enzyme determine d on SDS-polyacrylamide gel electrophoresis was 76 kDa.