Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.99.6 (sialyltransferase)
 1,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CMP-sialic acid:GM3 sialyltransferase (GD3 synthase; EC 2.4.99.8) was characterized in a membrane-enriched preparation (P2 pellet) from mouse embryos at embryonic day 12 (E-12). Gangliosides GD3 and GM3 were the major radiolabeled products of the reaction. Optimum GD3 synthase activity was obtained at pH 6.0 using 0.1% detergent Triton CF-54. The Km values for GM3 and CMP-sialic acid were 55 and 80 microM, respectively. The Vmax value was calculated as 622 pmol/mg protein/hr. Ganglioside GD3, as end product, induced a two-step reduction of enzyme activity in the range of concentrations from 0 to 34 microM (40%) and from 150 to 300 microM (65%). The rate of GD3 formation was similar in whole embryos and in embryo head and body regions. GD3 synthase activity in tw1/tw1 mutant mouse embryos, which express defects in neuronal differentiation, was only 40% of that in normal wild-type (+/+) embryos. Enzyme activity in heterozygous (+/twl) embryos was similar to that in +/+ embryos. These findings suggest that the reduced GD3 synthase activity in the mutants might arise as a consequence of failed nervous system development and might reflect a secondary rather than a primary effect of the mutation.
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PMID:Ganglioside GD3 biosynthesis in normal and mutant mouse embryos. 182 26

Sialidase and sialyltransferase activities were studied in JB6 mouse epidermal cells before and after exposure to phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), which irreversibly induces anchorage-independent growth and tumorigenicity. JB6 cells exhibited sialidase activities toward 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-NeuAc) and gangliosides at pH 4.5 in the particulate fraction but apparently not in the cytosol at pH 4.5 or 6.0. In JB6 cells exposed to TPA and in the anchorage-independent transformants, the sialidase activity toward 4MU-NeuAc was decreased and the activity toward gangliosides was increased compared with those in untreated JB6 cells. Immunological analysis with antisera against membrane-associated sialidases I and II revealed that plasma membrane-associated sialidase I was increased and lysosomal membrane-associated sialidase II was decreased under these conditions. TPA treatment also affected the sialyltransferase activities of JB6 cells: and elevation of the transfer activities toward asialo-orosomucoid and asialo-porcine submaxillary mucin but a reduction of GM3 and GD3 synthase activities were observed on exposure to TPA and in cells transformed by TPA to retain anchorage-independency. These results suggest that an increase in sialic acid bound to glycoproteins and a decrease in that bound to glycolipids may occur in JB6 cells exposed to TPA and in the anchorage-independent transformants.
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PMID:Tumor-promoting phorbol ester induces alterations of sialidase and sialyltransferase activities of JB6 cells. 212 97

A CMP-sialic acid: GM3 sialyltransferase (GD3 synthase) and a CMP-sialic acid: LacCer sialyltransferase (GM3 synthase) have been purified 10,000- and 3,000-fold, respectively, from the Triton X-100 extract of rat brain. The two enzymes were purified and resolved by affinity chromatography on two successive CDP-Sepharose columns by NaCl gradient elution. Final purification of GD3 synthase was achieved by specific elution from a 'GM3 acid'-Sepharose column with buffer containing GM3. Sodium dodecylsulfate-gel electrophoresis of GD3 synthase revealed a single major protein band with an apparent molecular weight of 55,000.
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PMID:Purification to homogeneity of GD3 synthase and partial purification of GM3 synthase from rat brain. 230 11

A simple and rapid procedure using anion exchange chromatography was established for determinations of the activity of ganglioside GD3 synthase (CMP-NeuAc: GM3, alpha 2----8 sialyltransferase) which catalyzes the conversion of ganglioside GM3 to GD3. The procedure was applicable for determination of activity of other ganglioside synthases. With the use of this procedure and GM3-acid affinity chromatography, the GD3 synthase was partially purified about 80 fold from a Lubrol PX extract of rat liver Golgi apparatus. The enzyme obtained had a pll optimum of 6.4. The preferred acceptors of the enzyme was GM3 containing N-acetylneuraminic acid (GM3 (NeuAc)) and GM3 containing N-glycolylneuraminic acid (GM3 (NeuGc)), with 2-fold higher V max in the latter than in the former. The Km values for GM3 (NeuAc), GM3 (NeuGc) and CMPNeuAc were 0.7 mM, 0.11 mM and 75 microM, respectively. The reaction product was identified as GD3 by thin layer chromatography. As to detergent, this enzyme showed maximum activity in 0.3% of Triton CF-54. The synthase did not require divalent cations for the activity, but rather Zn2+ and Cd2+ at 5 mM completely inhibited the enzyme activity. Cytidine nucleotides were strong inhibitors. The product, GD3, at 2 mM inhibited 30% the enzyme activity. The activity level of the GD3 synthase of rat liver was markedly different in rat strains, WKAH and TO strains being highest among eight strains examined. Male rat exhibited higher level than female. The synthase activity in rat liver was high at neonatal stage and decreased gradually thereafter.
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PMID:[A study of partial purification of ganglioside GD3 synthase and its enzymatic properties]. 232 44

Human melanoma cells express high levels of GM3 and GD3 gangliosides whereas normal melanocytes have only low levels of GD3 but maintain their expression of GM3. In order to understand the basis for this difference, the levels of the sialyltransferase that converts GM3 to GD3 (CMP-N-acetylneuraminic acid:GM3 sialyltransferase or GD3 synthase, EC 2.4.99.8) were analyzed in melanoma and other cell lines. Enzyme levels were determined in vitro using membrane preparations and measuring the addition of [14C]-N-acetylneuraminic acid from CMP-[14C]-N-acetylneuraminic acid to GM3 in the presence of Triton CF-54. Sialyltransferase levels in 44 human cancer cell lines (including melanoma, neuroblastoma, astrocytoma, various carcinomas, and leukemias) and cultures of normal melanocytes and kidney epithelial cells were compared, and the products were identified by thin layer chromatography and fluorography. Melanoma cell lines exhibited the highest levels of incorporation and GD3 was found to be the major product. GM3 was also formed, apparently from endogenous lactosylceramide. Very low levels of GD3 synthase were found in normal melanocytes. Neuroblastoma and some astrocytoma cell lines also had significant levels of GD3 synthase. Some other cell lines incorporated high levels of radioactivity but the products did not correspond to GD3 and the major product was usually GM3. In general the levels of GD3 synthase correlated with the expression of GD3 in the various cell types. These results point to higher levels of GD3 synthase being directly responsible for the enhanced expression of GD3 in melanoma.
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PMID:Sialyltransferase levels and ganglioside expression in melanoma and other cultured human cancer cells. 280 71

Three sialyltransferase activities involved in ganglioside biosynthesis were studied in Golgi-enriched preparations of rat liver: the formation of GM3, GD3 and GD1a. The conditions for the quantitative assays of these enzymatic reactions were standardized and optimized, with Triton X-100 being used as detergent. The apparent Km values of each sialyltransferase for N-acetyl-2-(5'-cytidylyl)neuraminic acid (1.5 mM with GM3 synthase, 0.2 mM with GD3 synthase, and 0.5 mM with GD1a synthase) and the respective glycolipid substrates (0.08 mM for lactosylceramide, 0.1 mM for GM3, and 0.5 mM for GM1) were determined. Competition experiments showed that the three sialyltransferase activities are three individual catalytic entities. Moreover, evidence was found that product inhibition may play a role in the regulation of the activity of sialyltransferases.
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PMID:Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases. 376 20

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 micrograms Golgi protein and 1 nmol UDP-GalNAc, optimal stimulation of GM2 synthase was obtained with 20 micrograms of phosphatidylglycerol and 7.5 nmol of the lipid acceptor GM3. Under the same experimental conditions this stimulation exceeds (by about 40%) that obtained with optimal amount (200 micrograms) 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 micrograms tunicamycin and more than 85% by 10 micrograms tunicamycin, per 50 micrograms Golgi membrane protein. The inhibition was stronger on GM1 synthesis: 85% with 2.5 micrograms 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. 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-mediated transport, e.g. of nucleotide sugars, across Golgi vesicles.
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PMID:Ganglioside biosynthesis in Golgi apparatus of rat liver. Stimulation by phosphatidylglycerol and inhibition by tunicamycin. 686 62

CMP-NAcNeu:GM3 ganglioside sialyltransferase (GD3 synthase) was characterized with respect to regulation of activity by nucleotides and compared in this regard with other sialyltransferases of ganglioside biosynthesis. Nucleotides preferentially inhibited the activity of GD3 synthase. Di- and trinucleotides inhibited most strongly and cytidine nucleotides were the most inhibitory class. The mode of inhibition by CMP (competitive or noncompetitive) varied with storage conditions of Golgi apparatus membranes; CMP inhibition was decreased during a series of consecutive freeze-thawings of membranes. Also, GD3 synthase inhibition by CDP was only partially relieved by excess Mg2+. With lactosylceramide as the in vitro precursor, synthesis of GM3 was always less inhibited by cytidine nucleotides than was that of GD3 and GT3. An 8-fold reduction in the ratio GD3/GM3 in the reaction products was obtained at 1.5 mM CTP. Separate incubations for the sialylation of GM3 or GM1 showed cytidine nucleotides increased synthesis of GD1a relative to GD3 by 3.5-fold.
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PMID:Ganglioside biosynthesis in rat liver: alteration of sialyltransferase activities by nucleotides. 740 17

Human fibroblast cell line WI-38 cultured in vitro was treated with a human recombinant IL-4 at concentrations of 1 to 100 U/ml to examine the alteration of glycosphingolipid (GSL) expression of the cells. Neutral GSL of non-treated WI-38 cells consisted of CMH (GlcCer), CDH, CTH, and Gb4Cer; CMH and CTH were the major components. The acidic GSL were composed of GM3 as the predominant component and other minor gangliosides including GD3. The neutral GSLs did not change in profile during the treatment with IL-4, while the acidic GSLs showed a prominent change, an increase of GD3 content. The increase of GD3 was detectable with IL-4 concentrations over 1 U/ml, and reached a plateau at 10 U/ml, where the amount of GD3 was almost equal to that of GM3. The GD3 increase occurred at 24 h after the IL-4 treatment, and lasted for at least 96 h, as long as IL-4 remained present in the culture media. The GD3 synthase (sialyltransferase) level was found to be increased in an IL-4 dose-dependent manner. IL-4 did not influence the growth or morphological appearance of WI-38 cells. The results demonstrate a novel biological effect of IL-4, modulating GSL in non-hematopoietic cells.
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PMID:Interleukin 4 enhances ganglioside GD3 expression on the human fibroblast cell line WI-38. 760 18

We have detected sialyltransferase activity of recombinant mouse STX, which was cloned from rat brain as a new member of the sialyltransferase family, but sialyltransferase activity of which had not been detected previously [Livingston and Paulson, J. Biol. Chem. (1993) 268, 11504-11507]. The activity of mouse STX was specific toward sialylated glycoproteins. N-Glycanase treatment and linkage-specific sialidase treatment of glycoproteins revealed that STX transfers sialic acids through alpha 2,8-linkages to only N-linked oligosaccharides of glycoproteins. However, polymerase activity for polysialic acid synthesis was not detected for this sialyltransferase. Since this alpha 2,8-sialyltransferase gene is highly restricted in fetal and newborn brain, it may be involved in the polysialylation of glycoproteins, especially of N-CAM.
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PMID:Enzymatic activity of a developmentally regulated member of the sialyltransferase family (STX): evidence for alpha 2,8-sialyltransferase activity toward N-linked oligosaccharides. 787 91


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