Characterization of a CMPNeuAc: lactosylceramide alpha 2----3sialyltransferase from rainbow trout hepatoma (RTH-149) cells.
1. The rainbow trout (Oncorhynchus mykiss) CMPNeuAc:lactosylceramide alpha 2----3sialytransferase enzyme from RTH-149 cells has been characterized. 2. Transfer of sialic acid to lactosylceramide was optimal at a pH of 5.9, temperature of 25 degrees C, and in the pressure of 0.3% CF-54, 10 mM Mn2+, 0.1 M sodium cacodylate, and 2 mM ATP. 3. Golgi-rich membrane fractions of RTH-149 cells were found to be enriched in sialidase activity and as such the addition of 40 microM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was necessary to assay alpha 2----3sialyltransferase activity optimally. 4. Apparent Km for donor (CMPNeuAc) and acceptor (lactosylceramide) were found to be 243 microM and 34 microM, respectively. 5. The alpha 2----3sialyltransferase characterized was found to be primarily specific for lactosylceramide though minor activity with other glycolipid acceptors was observed. 6. The presence of another sialyltransferase with differing substrate specificity was noted. 7. Properties of this enzyme, compared to analogous mammalian enzymes, are discussed.
Ostrander
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Pacific Northwest Research Foundation, Seattle, WA 98122.
Holmes
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D000818:Animals; D015703:Antigens, CD; D002240:Carbohydrate Sequence; D003902:Detergents / Q000494:pharmacology; D006028:Glycosphingolipids / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007790:Lactosylceramides; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014337:Trout; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D003902:Detergents; D006028:Glycosphingolipids; D007790:Lactosylceramides; D012794:Sialic Acids; C009744:CDw17 antigen; D012799:Sialyltransferases; C021343:haematoside synthetase; D019158:N-Acetylneuraminic Acid