Purification and characterization of UDP-N-acetylgalactosamine GM3/GD3 N-acetylgalactosaminyltransferase from mouse liver.

Abstract

A UDP-N-acetylgalactosamine: Sia alpha 2-3Gal beta 1-4Glc beta 1-/Sia alpha 2-8Sia alpha 2-3Gal beta 1-4Glc beta 1-1ceramide N-acetylgalactosaminyltransferase has been purified to apparent homogeneity from mouse liver. The purification procedure involved differential centrifugation for preparation of Golgi membranes, extraction of the enzyme with Triton X-100, and sequential chromatography on phosphocellulose, UDP-aldehyde adipic acid hydrazone agarose, UDP-hexanolamine-Sepharose, CM-Sepharose, and DEAE-Sepharose. At the phosphocellulose column chromatography step, the recovery of the enzyme activity was less than 25%, but it was enhanced up to 70% when the enzyme assay was performed in the presence of the flow-through fraction from the phosphocellulose column. With this assay, the enzyme activity was found to be quantitatively recovered during all the column chromatographies, the enzyme finally being purified 171,000-fold with a specific activity of 3.6 mumol/min/mg protein. The apparent molecular mass of the purified enzyme is 65,000 daltons. The enzyme exhibits a pH optimum of 7.5-7.9 and requires 2.5-10 mM Mn2+ for the maximal activity. The Km value for UDP-N-acetylgalactosamine is 7 microM. Among the glycolipids tested as acceptor substrates, NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide, NeuAc alpha 2-3 Gal beta 1-4Glc beta 1-1ceramide, NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide, and NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide are good ones, the Km values for them being 160, 2,100, 27, and 350 microM, respectively, but sialyllactose, NeuAc alpha 2-3Gal beta 1-4Glc, is not. This suggests that the enzyme recognizes not only the oligosaccharide portion but also the ceramide of gangliosides.