Biosynthesis in vitro of sialosylgalactosyldiacylglycerol by mouse brain microsomes.

A sialyltransferase enzyme, present in the microsomal fraction of mouse brain, catalyzes the synthesis in vitro of a lipid, characterized as 1,2-diacyl-3-beta-D-galactosyl (3 comes from 2 N-acetylneuraminosyl)-sn-glycerol, (sialosylgalactosyldiacylglycerol) from 1,2-diacyl-3-beta-D-galactosyl-sn-glycerol (galactosyldiacylglycerol) and cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NeuNAc). The enzymatic activity increases proportionally, over a given range, with increasing concentrations of both substrates and of enzyme. The apparent Km of the enzyme for galactosyldiacylglycerol is 130 microM, and for CMP-NeuNAc, 780 microM. The reaction proceeds optimally at pH 6.2. The product of the enzymatic reaction was characterized as a lipid which contained galactosyldiacylglycerol and N-acetylneuraminic acid. 14C-labeled lipid, synthesized from [14C]N-acetylneuraminic acid, and 3H-labeled lipid, synthesized from [3H]galactosyldiacylglycerol, ran with identical RF values when chromatographed on thin layers of silica gel. The water-soluble products, obtained by mild alkaline deacylation of these two labeled lipids, migrated the same when electrophoresed on paper. The ratio 14C/3H was calculated as 0.83 for doubly labeled lipid, [14C]sialosyl-[3H]galactosyldiacylglycerol. Degradation of this doubly labeled lipid by mild alkaline deacylation, followed by mild acid hydrolysis, yielded products that cochromatographed with standards galactosylglycerol and N-acetylneuraminic acid. Analysis of the products resulting from periodate oxidation of the 3H-labeled lipid demonstrated that the N-acetylneuraminic acid is linked to carbon 3 of the galactose.