Golgi vesicles were isolated and purified from rat liver, in which the specific activities of glycosyltransferases (e.g. GM3 : CMP-NeuAc sialyltransferase, GD3 synthase; G M 3 : UDP-GalNAc galactosaminyltransferase, GM2 synthase) were 50 -60-times enriched relative to microsonies or total homogenate. Synthesis of gangliosides GM2 and GhIl in such Golgi vesicles is, in the abscnce of any detergents, stimulated 6-fold and 20-fold respectively by phosphatidylglycerol. Other phospholipids like phosphatidylethanolamine and phosphatidylserine are also significantly stimulatory. With 50 pg Golgi protein and 1 nmol UDP-GalNAc, optimal stimulation of GMz synthase was obtained with 20 pg of phosphatidylglycerol and 7.5 nmol of the lipid acceptor GM3. Under the same experimental conditions this stimulation exceeds (by about 40 x) that obtained with optimal amount (200 pg) 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 ME"+ or M n 2 + , indicating that facilitation by phospholipid of CiM3 transport into Golgi vesiclcs was not the basis of stimulation of GMz synthesis.Tunicamycin inhibits the synthesis of gangliosides G M 2 and GMI in isolated Golgi vesicles, but only in the absence of detergents. In the prescnce of phosphatidylglycerol, GMz synthesis, for example, was inhibited by 60% by 2 pg tunicamycin and more than 85"/:, by 10 pg tunicamycin, per 50 pg Golgi membrane protein. The inhibition was stronger on GMl synthesis: 85 ; $ with 2.5 pg 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 incrcasingly 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. All these results indicate that phosphatidylglycerol does not stimulate. and tunicamycin does not inhibit, the transferases themselves; rather, the two opposing effects might relate to carrier-mediatcd transport, e.g. of nucleotide sugars, across Ciolgi vesicles.The understanding ol' the mechanism of ganglioside biosynthesis in Golgi apparatus is restricted by the lack orknowledge about how the nucleotide sugars (cg. UDP-Gal, UDPAbhrrviutims. 1JDP-Gal, uridinc 5-diphosphogalactose; UDPGalNAc, uridine 5'-diphospho-N-acetylgalactosamine; UDP-GlcNAc, uridine 5'-N-acctylglucosaiiiine; GDP-Fuc, guanosine 5'-diphosphofucose: CDP-choline, cytidine 5'-diphosphocholine; CMP-NeuAc, cytidine 5'-monophospho-N-acetylneuraminic acid : GlcNAc-1 -1 " . N-acetylz-o-glucosamine-I-phosphate; GalNAc, N-acetyl-a-u-palactosamine; Dol-P, dolichyl monophosphate; Dol-P-P-GlcNAc. N-acctylglucosaminyldiphosphoryldolichol; octylglucoside; l-O-I1-octyl-/i-u-glucopyranosidc; Neu...
The synthesis of ganglioside GM1 in intact rat liver Golgi-derived vesicles is stimulated by phosphatidylglycerol as much (about 20-fold) as by Triton X-100. The antibiotic tunicamycin inhibits strongly the synthesis, in the presence as well as in the absence of the phospholipid, but has no effect when Golgi membranes are solubilized with detergent. In Pronase-treated Golgi vesicles, which retain fill enzyme activity, both phospholipid dependence and tunicamycin inhibition of the synthesis disappear completely. When freshly prepared Golgi vesicles are incubated with 125 IAM UDP-[3H]Gal for 10 min at 30%C, the nucleotide sugar is found to be transported into the vesicles at an approximate rate of about 85 pmol/mg of protein per min, 92% of which remains firmly bound to the membrane. Tunicamycin inhibits this transport in a concentration-dependent manner. These results indicate the existence of carrier proteins in rat liver Golgi vesicles, which mediate the transport of the sugar nucleotide UDP-Gal, and that the carriers face the cytoplasmic side of the vesicles. The results also show that, although the mechanism of phosphatidylglycerolinduced stimulation of the synthetic activity remains unclear, tunicamycin inhibits ganglioside biosynthesis by blockdng the transport of the nucleotide sugar and not by inhibiting the transferase directly.One prerequisite step in ganglioside biosynthesis in the Golgi apparatus is the entrance of the sugar nucleotides from the outer side into the vesicles. This is because the sugar nucleotides are synthesized almost exclusively in the cytoplasm (1, 2), whereas the glycosyltransferases that catalyze the transfer of sugar residues to glycolipid (and glycoprotein) acceptors are believed to face the Golgi lumen (3-6). Although the mechanism by which the activated sugars enter the lumen of the Golgi vesicles is unclear, recent studies of Sommers and Hirschberg (7) indicate the transport of CMP-NeuAc and GDP-Fuc to be mediated by carrier proteins.In vitro studies reported thus far on ganglioside biosynthesis (8)(9)(10)(11)(12)(13)(14) have involved the use of detergents for solubilizing the membranes, so that the sugar nucleotides and other reaction components are directly available to the transferases. These studies, although providing valuable information on the functional properties of the enzymes, could not detect any carriermediated transport of the activated sugars. We developed a system in which full enzyme activity could be obtained, without disrupting the membrane vesicles; in Golgi vesicles isolated from rat liver, phosphatidylglycerol stimulates more strongly than octylglucoside (detergent) the synthesis of ganglioside GM2 (15).Tunicamycin, the antibiotic known to be an inhibitor of glycoprotein biosynthesis in microsomes (see ref. 16 for review), also inhibits the synthesis of ganglioside GM2 in Golgi membranes (15) and ganglioside biosynthesis in neuroblastoma cells (17).In this report, we present data on the stimulation by phosphatidylglycerol and inhibition by tuni...
Malnutrition in mice from birth resulted in myelin of brain having higher than normal molar proportions of cholesterol and phospholipids relative to a molar unit of cerebroside + sulphatide. This was found at all ages between 20 and 60 days, and the molar ratio of these lipids in older animals was comparable to that in the younger controls. The phospholipid and the ganglioside patterns were also immature for age. The phospholipid composition was characterized by lower molar proportions of ethanolamine phosphoglyceride (EPG) and sphingomyelin (SPh) and higher proportion of choline phosphoglyceride (CPG), and the ganglioside pattern was characterized by higher molar proportions of the disialogangliosides GD1a and GD1b and markedly lower proportion of the monosialoganglioside GM1. Malnutrition imposed from 30 days of age did not affect the contents of the major lipids (and so their molar ratio), but within the phospholipids there was a small but significant deficit of SPh, which was compensated by a higher content of CPG. The ganglioside pattern was as if the animals were malnourished from birth. Nutritional rehabilitation up to 60 days of age subsequent to malnutrition for the first 30 days fully corrected the ganglioside pattern, but not the molar ratio, of the major lipids (because of persistent deficit in cerebroside + sulphatide) and the composition of the phospholipids (because of small but significant deficit of SPh). The results indicate that malnutrition instituted at any time during the entire programme of myelination can affect one or other aspect of myelin development, and nutritional rehabilitation of animals malnourished in early life cannot fully correct this developmental gap.
The increase observed in the amount of the disialoganglioside GDlof the rat cerebrum during development between 21 and 81 days of age accounted for nearly 40% of the overall increase in total ganglioside in the tissue during the same period. Subcellular fractionation showed the microsomal fraction to contribute by far the most towards this increase in Cerebral ganglioside GDla. It is suggested that microsomal ganglioside GDla may serve as a marker for dendritic arborization in the rat cerebrum.
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