Ganglioside Biosynthesis in Golgi Apparatus of Rat Liver

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Abstract
Golgi vesicles were isolated and purified from rat liver, in which the specific activities of glycosyltransferases (e.g., GM3: CMP-NeuAc sialyltransferase, GD3 synthase; GM3: UDP-GalNAc galactosaminyltransferase, GM2 synthase) were 50-60-times enriched relative to microsomes or total homogenate. Synthesis of gangliosides GM2 and GM1 in such Golgi vesicles is, in the absence of any detergents, stimulated 6-fold and 20-fold, respectively, by phosphatidylglycerol. Other phospholipids like phosphatidylethanolamine and phosphatidylserine are also significantly stimulatory. With 50 .mu.g Golgi protein and 1 nmol UDP-GalNAc, optimal stimulation of GM2 synthase was obtained with 20 .mu.g of phosphatidylglycerol and 7.5 nmol of the lipid acceptor GM3. Under the same experimental conditions this stimulation exceeds (by .apprx. 40%) that obtained with optimal amount (200 .mu.g) of the detergent octylglucoside. Phosphatidylglycerol, on the other hand, has virtually no stimulatory activity on the synthesis of ganglioside GD3 either in the presence of Mg2+ or MN2+, indicating that facilitation by phospholipid of GM3 transport into Golgi vesicles was not the basis of stimulation of GM2 synthesis. Tunicamycin inhibits the synthesis of gangliosides GM2 and GM1 in isolated Golgi vesicles, but only in the absence of detergents. In the presence of phosphatidylglycerol, GM2 synthesis, for example, was inhibited by 60% by 2 .mu.g tunicamycin and > 85% by 10 .mu.g tunicamycin, per 50 .mu.g Golgi membrane protein. The inhibition was stronger on GM1 syntehsis: 85% with 2.5 .mu.g of the antibiotic. The dependence on phosphatidylglycerol and the degree of inhibition by tunicamycin of the synthetic activities are strictly dependent on the intactness of the Golgi vesicles: both phenomena become increasingly less evident when the vesicles are pelleted, and frozen and thawed several times, and completely disappear when the vesicles are solubilized by detergents or disrupted by ultrasonication. Furthermore, tunicamycin inhibition is reversible by increased concentration of phosphatidylglycerol. Phosphatidylglycerol does not stimulate, and tunicamycin does not inhibit, the transferases themselves; rather, the 2 opposing effects might relate to carrier-mediated transport, e.g., of nucleotide sugars, across Golgi vesicles.

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