Glycolipid sialosyltransferase activity in synaptosomes exhibits a product specificity for (2-8)disialosyl lactosyl ceramide (ganglioside GD3).
Intact synaptosomes prepared from 28-day-old rat brains were incubated with CMP-N-acetyl-(14C) neuraminic acid in Krebs-Henseleit buffer in an atmosphere of 95% O2: 5% CO2, at 37 degrees C. The activity of CMP-NANA:ganglioside sialosyltransferase using endogenous acceptors was 0.84 pmoles NANA transferred/mg synaptosomal protein/hr. Analysis of the distribution of labeled sialic acid revealed that GD3 ganglioside (alpha 2----8 disialosyl, alpha 2----3 galactosyl, beta 1----4 glucosyl, beta 1----1-ceramide) was the major product in the membrane carrying 32% of the total lipid bound label. Treatment of the reaction products with Clostridium neuraminidase liberated labeled sialic acid from GD3 and yielded labeled GM3, then unlabeled lactosyl ceramide. Lac-cer and GM3 are present in small amounts in synaptosomes, and GD3 represents less than 2% of the total ganglioside. Our findings indicate that the sialosyltransferase activity of synaptosomes exhibits a preferential product specificity for the small pool of synaptosomal membrane GD3 ganglioside that may be formed in situ, via sialosylation of its precursor (GM3 or lactosyl ceramide) which pre-exists in the synaptosomal plasma membrane. The second major labeled product quantitatively was GD1a whose precursor substrate, GM1, is quite abundant in the membrane, so that the conversion rate of GM1 to GD1a was low in comparison with GD3 formation. Sialosylation of other synaptosomal membrane gangliosides was negligible.
Durrie
R
R
Division of Molecular Biology and Neuroregeneration, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962.
Saito
M
M
Rosenberg
A
A
D000818:Animals; D001923:Brain Chemistry; D002462:Cell Membrane / Q000201:enzymology; D002795:Choline O-Acetyltransferase / Q000032:analysis; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000032:analysis* / Q000493:pharmacokinetics; D005625:Frontal Lobe / Q000166:cytology / Q000201:enzymology*; D005732:Gangliosides / Q000378:metabolism*; D009439:Neuraminidase; D009708:Nucleotidases / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D012794:Sialic Acids / Q000032:analysis*; D013379:Substrate Specificity; D013574:Synaptosomes / Q000201:enzymology*
D005732:Gangliosides; D012794:Sialic Acids; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; C026226:ganglioside, GD3; D002795:Choline O-Acetyltransferase; D009708:Nucleotidases; D009439:Neuraminidase