Ganglioside biosynthesis in mouse embryos: sialyltransferase IV and the asialo pathway.
The in vitro activity of sialyltransferase IV (SAT-IV), which catalyzes the transfer of sialic acid to the terminal galactose of different gangliotetraosylceramides (GA1, GM1a and GD1b), was examined in membrane-enriched preparations from mouse embryos at embryonic day 12 (E-12). Gangliosides GD1a and GT1b were the only reaction products using GM1a and GD1b as substrates, respectively. The Km values for GM1a and GD1b were 53 microM and 42 microM, respectively. Competitive inhibition experiments showed that the same enzyme (SAT-IV) catalyzed sialic acid transfer to the terminal galactose residues of both GM1a and GD1b. Two labeled ganglioside products were obtained, however, using GA1 as a substrate. One product was identified as ganglioside GM1b and the enzymatic reaction for its formation was maximal at pH 6.0, similar to that for GD1a and GT1b formation. The second product, synthesized by a different sialyltransferase, was identified as GD1 alpha based on results from TLC immunostaining, neuraminidase digestion, and periodate oxidation-borohydride reduction. The pH dependence curve for GD1 alpha formation had a different shape than that for GM1b formation with a maximum at pH 6.3. GD1 alpha is apparently synthesized from GM1b by an endosialyltransferase that catalyzes the transfer of a second sialic acid to the internal N-acetylgalactosamine of GM1b. The formation of both GM1b and GD1 alpha was linear over protein concentration. The ratio of GD1 alpha/GM1b formation varied from 0.25 to 1.20 depending on the GA1 substrate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Seyfried
T N
TN
Department of Biology, Boston College, Chestnut Hill, MA 02167.
Novikov
A M
AM
Irvine
R A
RA
Brigande
J V
JV
D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004622:Embryo, Mammalian / Q000378:metabolism*; D005260:Female; D005677:G(M1) Ganglioside / Q000031:analogs & derivatives / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005732:Gangliosides / Q000096:biosynthesis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008809:Mice, Inbred C3H; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005732:Gangliosides; D012794:Sialic Acids; C021298:ganglioside, GD1a; C033851:ganglioside, GD1b; D005677:G(M1) Ganglioside; C029624:ganglioside M1b; C016481:trisialoganglioside GT1; C018835:asialo GM1 ganglioside; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D005690:Galactose