Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Two different sialyltransferases (EC 2.4.99.1) have been resolved from Triton X-100 extracts of porcine submaxillary glands by affinity chromatography on CDP-hexanolamine agarose. The predominant sialyltransferase of this tissue, a CMP-N-acetylneuraminate: alpha-D-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase, has been obtained in a partially purified and stable form. A less abundant but highly active enzyme, a CMP-N-acetylneuraminate: beta-D-galactoside alpha2 leads to 3 sialyltransferase, was purified over 90,000-fold to homogeneity. Chromatography of the latter enzyme on Sephadex G-200 separated two noninterconverting forms, designated A and B, with Stokes radii of 51 A and 31 A, respectively. Both forms have equal specific activity toward lactose and contain a single polypeptide with a molecular weight of about 50,000 as estimated by gel electrophoresis. Form A appears to bind 1.18 g of Triton X-100 per g of protein, or nearly an entire detergent micelle per polypeptide, while Form B binds little or no detergent. The enzymatic properties of both forms are similar (Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) J. Biol. Chem. 254, 4444-4451) supporting the conclusion that Form A may represent the native sialyltransferase with an intact membrane-binding site, and Form B may be a large proteolytic fragment of Form A.
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PMID:Purification to homogeneity of a beta-galactoside alpha2 leads to 3 sialyltransferase and partial purification of an alpha-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase from porcine submaxillary glands. 43 96

By means of affinity chromatography on CDP-hexanolamine-agarose, a CMP-N-acetylneuraminate: alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase (EC 2.4.99.1) has been purified 117,000-fold to homogeneity from Triton X-100 extracts of porcine submaxillary glands. The enzyme consists of several electrophoretic forms that can be partially resolved by chromatography on Sephadex G-200, the largest of which has a molecular weight of approximately 160,000 as estimated by sodium dodecyl sulfate-gel electrophoresis. Periodate oxidation studies show that the linkage formed by this enzyme with ovine submaxillary asialo-mucin as the acceptor substrate is NeuAc alpha 2 leads to 6GalNAc alpha 1 leads to O-Thr/Ser. On the basis of initial rate studies and the patterns of inhibition observed with alternate acceptor substrates, the transferase is proposed to have either a random equilibrium kinetic mechanism or an ordered steady state mechanism with the acceptor substrate binding first. Among a wide variety of oligosaccharides, glycoproteins, and simple glycosides (including p-nitrophenyl-alpha-N-acetylgalactosaminide), the only acceptor substrates for this enzyme are those glycoproteins containing the structure, R leads to 3GalNAc alpha 1 leads to O-Thr/Ser, where R may be H or a beta-galactoside.
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PMID:Purification to homogeneity and enzymatic characterization of an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase from porcine submaxillary glands. 44 88

CDP-hexanolamine agarose was used as an affinity adsorbent to purify a CMP-N-acetylneuraminate: beta-D-galactosyl-glycoprotein N-acetylneuraminyltransferase (EC 2.4.99.1) from bovine colostrum. Upon binding of the enzyme to the adsorbent, elution is achieved either nonspecifically, with 0.5 to 1.0 M sodium chloride, or specifically, with CDP. A highly purified sialyltransferase is obtained with a specific activity 440,000 times that of whole colostrum. Fractionation of the purified enzyme by gel filtration gives two species with different molecular weights but equal specific activities toward asialo-alpha1-acid glycoprotein (26.0 to 28.0 micronmol/min/mg of enzyme). The molecular weights of these two forms are about 56,000 and 43,000 as judged by sodium doedcyl sulfate-gel electrophoresis, sedimentation equilibrium, and gel filtration. The catalytic properties of both forms have been examined (Paulson, J. C., Rearick, J. I., and Hill, R. L. (1977) J. Biol. Chem. 252, 2363-2371). It is concluded that the lower molecular weight form may be a partially degraded species of the enzyme of higher molecular weight.
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PMID:Purification of a sialyltransferase from bovine colostrum by affinity chromatography on CDP-agarose. 84 32

A CMP-NeuAc:GM1 alpha 2-3 sialyltransferase (GD1a synthase, 2.4.99.2) has been purified from the Triton extract of rat brain. The enzyme was purified and resolved by affinity chromatography on CDP-Sepharose column by a linear NaCl gradient elution. Final purification was achieved by elution from a 'GM1-acid'-Sepharose column. SDS-PAGE of the enzyme revealed a single protein band with an apparent Mr 44 kDa. It catalyzed specifically the sialylation of GD1b, GM1 and asialo-GM1. Enzyme products were identified by TLC in three different solvent systems. The Km value for GM1 was 7.5 x 10(-2) M, and for CMP-NeuAc it was 6.5 x 10(-5) M.
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PMID:Purification and characterization of CMP-NeuAc:GM1 (Gal beta 1-4GalNAc) alpha 2-3 sialyltransferase from rat brain. 226 6

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

The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.
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PMID:In vitro synthesis of colominic acid by membrane-bound sialyltransferase of Escherichia coli K-235. Kinetic properties of this enzyme and inhibition by CMP and other cytidine nucleotides. 264 17

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.
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PMID:Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta. 398 39

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma differ in morphology, agglutinability with concanavalin A, and xenotransplantability. Both cell lines contain a major mucin-type glycoprotein, but the MAT-C1 (xenotransplantable) subline contains a 3-fold-greater content of sialic acid on the glycoprotein than does the MAT-B1 (nonxenotransplantable) subline. The present work indicates that whole cells of both lines incorporate radioactivity from labeled CMP-sialic acid into a component which comigrates with the major glycoprotein by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and that label incorporated by MAT-B1 cells is released by alkaline-borohydride treatment. Sialyltransferase can be purified from 250- to 400-fold by chromatography of a Triton X-100 extract of microsomes on CDP-agarose. The purified fraction of both cell lines has a Km for CMP-sialic acid of 0.40 +/- 0.10 mM with asialofetuin as the acceptor, and gives 35 to 40% of the activity with the acceptor asialotransferrin as with asialofetuin. When assayed with a variety of acceptors, the MAT-C1 extract showed higher sialyltransferase activity at a pH below 6.5 than did the MAT-B1 extract. Analysis of the products following incubation with lactose yields only 3'-sialyllactose for both cell lines. The results indicate that the differences in MAT-B1 and MAT-C1 sialyltransferase when assayed with glycoprotein acceptors are not large enough to account for the differences in sialic acid content of the two cell lines.
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PMID:Sialyltransferase of the 13762 rat mammary ascites tumor cells. 669 99

Rat liver Golgi was found to contain a sialyltransferase activity which would convert lacto-N-tetraose (Gal beta 1 goes to 3GlcNAc beta 1 goes to 3Gal beta 1 goes to 4Glc) to LS-tetrasaccharide a (NeuAc alpha 2 goes to 3Gal beta 1 goes to 3GlcNAc beta 1 goes to 3Gal beta 1 goes to 4Glc). The enzyme has been partially purified by affinity chromatography on CDP-hexanolamine agarose. Of the glycoprotein substrates examined, it utilizes the Gal beta 1 goes to 3GlcNAc sequence found on the asparagine-linked oligosaccharides of prothrombin as its preferred acceptor substrate, and thus has been tentatively designated a Gal beta 1 goes to 3GlcNAc alpha 2 goes to 3 sialyltransferase. The partially purified enzyme has an acceptor specificity distinct from other purified mammalian sialyltransferases which synthesize the NeuAc alpha 2 goes to 3Gal beta 1 goes to 3 GalNAc and NeuAc alpha 2 goes to 6 GalNAc sequences common to oligosaccharides O-linked to threonine or serine and the NeuAc alpha 2 goes to 6Gal beta 1 goes to 4GlcNAc sequence found on oligosaccharides N-linked to asparagine.
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PMID:Identification of a Gal beta 1 goes to 3GlcNAc alpha 2 goes to 3 sialyltransferase in rat liver. 706 22

A Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferse and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase have been purified 23,000- and 860,000-fold to homogeneity from Triton CF-54 extracts of rat liver membranes. The two enzymes were concentrated by affinity chromatography on CDP-hexanolamine-agarose and resolved by NaCl gradient elution from the same adsorbent. Final purification of the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialytransferase, the most abundant enzyme, was achieved by specific elution from CDP-agarose with CDP. The Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase was also purified further by CDP elution from CDP-agarose, but final purification required affinity chromatography on an adsorbent prepared by coupling asialoprothrombin to cyanogen bromide-activated agarose. Asialoprothrombin contains the terminal sequence Gal beta 1 to 3GlcNAc on N-linked oligosaccharides and is the best acceptor substrate of the enzyme (Km congruent to 6 microM). The Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase was found to bind to asialoprothrombin-agarose in the presence of CDP and could be eluted with a solution containing 0.2 M lactose and no CDP. Sodium dodecyl sulfate-gel electrophoresis of the Gal beta 1 to 4GlcNAc alpha 2 to 6 and Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferases revealed a single major protein band for each enzyme with apparent molecular weights of 40,500 and 44,000, respectively. Rabbit antibodies raised to the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase inhibit its enzymatic activity greater than 99% but caused little or no inhibition of Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialytransferase. Moreover, the Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase quantitatively bound to a column containing antibody adsorbed to Protein A-agarose, while the Gal beta 1 to 3(4) GlcNAc alpha 2 to 3 sialyltransferase did not bind. This demonstrated that the two sialyltransferases are antigenically unrelated and formed the basis for removal of contaminating Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase from solutions of the Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase. Enzymatic characterization of the two sialyltransferases suggests that their major biological roles are in the terminal glycosylation of N-linked oligosaccharides of glycoproteins. (Weinstein, J., de Souza-e-Silva, U., and Paulson J. C. (1982) J. Biol. Chem. 257, 13845-13853. The alpha 2 to 6 sialyltransferase efficiently forms the NeuAc alpha 2 to 6Gal beta 1 to 4GlcNAc sequence, and the alpha 2 to 3 sialyltransferase forms the NeuAc alpha 2 to 3Gal beta 1 to 3GlcNAc and NeuAc alpha 2 to 3Ga; beta 1 to 4GlcNAc sequences.
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PMID:Purification of a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase to homogeneity from rat liver. 714 79


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