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:2.4.99.7 (sialyltransferase)
1,534 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper presents kinetic properties of the transfer of several synthetic 9-substituted sialic acid analogues onto N- or O-linked glycoprotein glycans by four purified mammalian sialyltransferases: Gal beta 1,4GlcNac alpha 2,6sialyltransferase, Gal beta-1,4(3)GlcNAc alpha 2,3-sialyltransferase, Gal beta 1,3GalNAc alpha 2,3sialyltransferase, and GalNAc alpha 2,6sialyltransferase. The substituents at C-9 of the sialic acid analogues introduce special biochemical characteristics: 9-Amino-NeuAc represents, up to the present, the first derivative that is resistant toward bacterial, viral, and mammalian sialidases but is transferred by a sialyltransferase. 9-Acetamido-NeuAc, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc differ in size and hydrophobic character from each other and from parent NeuAc. 9-Azido-NeuAc may be used to introduce a photoreactive label. The kinetic properties of the four sialyltransferases with regard to the donor CMP-glycosides differed distinctly depending on the structure of the substituent at C-9. CMP-9-amino-NeuAc was only accepted as donor substrate by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase (rat liver), but the Km value was 14-fold higher than that of parent CMP-NeuAc. In contrast, 9-azido-NeuAc was readily transferred by each of these four enzymes. 9-Acetamido-NeuAc, which is a receptor analogue for influenza C virus, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc were also accepted by each sialyltransferase, but incorporation values differed significantly depending on the enzyme used. For the first time, the resialylation of asialo-alpha 1-acid glycoprotein with 9-substituted sialic acid analogues by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase is demonstrated.
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PMID:Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases. 251 Aug 24

Sialyltransferase activity in normal human breast tissue and tumors was investigated with lactose, desialylated fetuin, and bovine submaxillary mucin as the acceptors. While microsomal preparations from the normal tissue showed little or no sialyltransferase activity toward these acceptors, tumors showed elevated enzymic activities. Tween-20 at 0.5% concentrations stimulated sialic acid transfer to all three acceptors. Another nonionic detergent, Triton X-100, stimulated asialo fetuin sialyltransferase activity while inhibiting activity toward asialo BSM and lactose. Interestingly, lysolecithin, a normal cellular constituent which possesses detergent properties also had an effect similar to that of Triton X-100. Thermal denaturation curves of enzymic activity toward asialo BSM, however, resembled those seen with asialo fetuin as the acceptor. Kinetic studies showed that at acceptor concentrations of 500 micrograms each, sialyl transfers to asialo fetuin, asialo BSM, and lactose showed apparent Km values of 50, 60, and 300 microM, respectively. At CMP-sialic acid concentrations of 300 microM, the Km values for the above acceptors were 25, 15, and 5000 microM.
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PMID:Some biochemical properties of human breast tumor sialyltransferase. 258 61

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

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

Sialyltransferase-1 activity was studied in cultured 12-18 human glioma cells. The apparent Km and Vmax with variable LacCer concentrations were 32 microM and 197 pmoles/mg protein/hr and with variable CMP-NeuAc concentrations were 172 microM and 877 pmoles/mg protein/hr., respectively. The pH optimum towards exogenous LacCer was 6.0 and towards endogenous acceptors was 6.2. The optimum protein:detergent ratio was 1:1. Human beta interferon (1000 units/ml medium) increased sialyltransferase-1 activity only slightly on a protein basis but increased it 47% on a per cell basis. These results demonstrate that one of the biochemical effects of beta-interferon on 12-18 human glioma cells is to stimulate ganglioside synthesis.
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PMID:Sialyltransferase-1 in a human malignant glioma cell line. Kinetic characteristics and effect of human interferon-beta. 285 19

The Ia+ B cell lymphoma, AKTB-1b, fails to stimulate thymic lymphocytes in a one-way mixed lymphocyte reaction unless pretreated with sialidase or inhibitors of N-linked oligosaccharide processing. A comparison of different sialidases and sialyltransferases suggests that the removal of only a subset of total surface sialic acid, rather than net desialylation of the cell surface, is required. Three sialidases were compared, including Vibrio cholerae (VC) and Clostridium perfringens (CP), which will cleave alpha 2-3, alpha 2-6, and alpha 2-8, sialic acid linkages, and Newcastle Disease virus (NDV), which will remove only alpha 2-3 and alpha 2-8 linked sialic acid. When treated with equivalent units of sialidase, CP-, VC-, and NDV-treated cells were 24-fold, sixfold, and threefold better stimulators than untreated cells. In contrast, VC released 1.3-fold and 2.5-fold more sialic acid per cell than did CP or NDV, respectively. Furthermore, VC was superior in reducing the levels of binding of the sialic acid-specific lectin, Limulus polyphemus agglutinin, in exposing Gal beta 1-3GalNAc and Gal beta 1-4GlcNAc residues, and in desialylating gangliosides. Two-dimensional gel analysis indicated that VC and CP were both equal and superior to NDV in the desialylation of iodinatable cell-surface proteins, including H-2Kk, I-A beta k, and a highly sialylated 65,000 dalton protein of unknown identity. Maximal resialylation of CP-treated cells with exogenously added CMP-NANA and either the alpha 2-3(Gal beta 1-3GalNAc) or alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase did not reduce the stimulatory capacity of these cells. However, resialylation of VC-treated cells with just CMP-NANA alone resulted in 49% reversal of their stimulatory capacity, and no additional reversal could be achieved with either of the sialyltransferases. Although the alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase was capable of adding back approximately 10% of the sialic acid removed, the endogenous activity added back approximately 0.1% of the total sialic acid removed. SDS-PAGE gels of the sialylated cells indicated that the exogenously added sialyltransferase labeled many different proteins, whereas the endogenous activity labeled far fewer proteins, predominantly in 46,000 and 25,000 m.w. range. Both the desialylation and resialylation data suggest that the sialidase-dependent stimulation is due to the desialylation of specific membrane structures. Together with previous studies, these data suggest that the sialic acids involved are probably alpha 2-6 linked to N-linked glycosyl moieties.
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PMID:Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction. 295 14

Purified lactotetraosylceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc1-Cer) was tested for its ability to accept [14C]sialic acid from CMP-[14C]sialic into monosialoganglioside fractions in the presence of membrane fractions purified from human colorectal carcinoma cells (SW1116). Membrane fractions were isolated by three different methods: sucrose density centrifugation, CMP-agarose gel column chromatography, and LcOse4 gel chromatography. We optimized the incubation conditions for detergent dependency (taurocholate), pH (6.3), and acceptor concentration. The sialyltransferase activity was dependent on membrane protein and linear for time up to at least 4 h. The LcOse4 affinity chromatography of the crude microsomal membrane pellet from these cells yielded a membrane fraction that was 136-fold enriched in LcOse4 acceptor specific activity compared to cell homogenates. The apparent Km for the sialyltransferase activity with LcOse4Cer acceptor in the presence of affinity-purified membranes was 20 microM and the Vmax was 7 pmol h-1 (100 micrograms of protein)-1. Acceptor capabilities of other core structures were 5-20-fold lower: LcOse4Cer much greater than GgOse4Cer greater than nLcOse4Cer much greater than GbOse4Cer. The enzymatic activity was purified further (900-fold) by a combination of LcOse4 and CMP affinity gels. SDS-PAGE electrophoresis of this material showed a major set of closely migrating bands of Mr 58,000-54,000 compared to authentic proteins, as well as a minor band at 27,000. We analyzed picomole quantities of the radioactive product by convenient controlled short-term hydrolyses with an endoglycoceramidase and sialidases (from four different sources) in comparison to sialylated tetrasaccharides of known structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of a CMP-sialic:LcOse4Cer sialyltransferase from human colorectal carcinoma cell membranes. 306 17

The viscosity of plasma and extracellular fluid has been shown to be a regulator of lipoprotein production both in cultured hepatocytes and in vivo. The possibility that this extracellular effect on cell function involves modulation of cell surface membrane components was examined. In the present work, we studied the effect of medium viscosity on liver cell gangliosides known to be involved in various membrane functions and to be located predominantly at the cell surface membrane. Cultivation of isolated hepatocytes as primary cultures markedly reduced the ganglioside content, but this reduction process was attenuated by increasing the viscosity of the culture medium. Elevation of extracellular fluid viscosity inhibited the degradation of the cell gangliosides and secretion of lysosomal enzymes involved in ganglioside degradation. The cellular activity of these enzymes as well as the activity of enzymes involved in ganglioside synthesis, CMP-NANA:GM1 sialyltransferase, CMP-NANAP:GM3 sialyltransferase and UDP-galactose:GD2 galactosyltransferase, were not affected by modulation of the extracellular medium viscosity. It is proposed that the modulation of cell ganglioside content by extracellular fluid viscosity is due to an effect on enzymes involved in ganglioside catabolism.
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PMID:Regulation of liver cell ganglioside composition by extracellular fluid viscosity. 309 15

To investigate the presence of glycosyltransferase activity at the apical surfaces of columnar cells in small intestine, CMP-[3H]-sialic acid was injected into the lumen of a ligated segment of rat jejunum; 5 min later the tissue was fixed and processed for light microscopic autoradiography. After a 3-6-month exposure, an autoradiographic reaction appeared over the microvillar surfaces of columnar cells, indicating the presence of surface sialyltransferase activity accompanied by endogenous acceptors. When CMP-[3H]-sialic acid was injected into the posterior chamber of rat eye or the lumen of mouse gallbladder, no autoradiographic reaction was observed at the surfaces of the cells facing these cavities. After injection of UDP-[3H]-galactose into the same three sites, an autoradiographic reaction was observed in the Golgi regions of the various epithelial cells, but not along their apical surfaces. Competition experiments using unlabeled galactose indicated that [3H]-galactose had been released from the nucleotide and had entered the cells to be incorporated into the Golgi apparatus.
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PMID:Autoradiographic demonstration of in vivo sialylation of endogenous acceptors at the microvillar surface of intestinal columnar cells after intraluminal administration of CMP-[3H]-sialic acid. 311 Feb 65

Recent reports have suggested that the majority of the molecular traffic through the Golgi apparatus is comprised of recycling, rather than newly synthesized, molecules. To evaluate the importance of this recycling pathway in greater detail, we examined the internalization and recycling of cell surface glycoproteins on EL-4 cells, a murine T-cell lymphoma, using sialic acids as covalent markers. Sialic acids were removed from the surface of living cells by exhaustive treatment with Vibrio cholerae sialidase at 4 degrees C and shown to be derived primarily from glycoproteins (93%), with only a small amount from glycolipids (7%). Cells were recultured at 37 degrees C over time and monitored for the resialylation of the cell surface using a sensitive high pressure liquid chromatography adaptation of the thiobarbituric acid assay for sialic acids. The return of sialic acid to the cell surface was found to be contingent upon de novo protein synthesis indicating that the bulk of plasma membrane sialoglycoconjugates do not recycle to an endogenous sialyltransferase-containing compartment for oligosaccharide reprocessing. Identical results were found for K562 cells, a human erythroleukemia cell line. The movement of specific glycoproteins was followed using the enzyme rat liver alpha 2-6Gal beta 1-4GlcNAc sialyltransferase together with CMP-[3H]NeuAc as an impermeant probe of the cell surface. Surface sialoglycoproteins were internalized slowly, a process unaffected by cycloheximide treatment. Only a few of these internalized glycoproteins were found to return to a trans-Golgi compartment followed by recycling to the cell surface. Taken together, these data indicate that the majority of replacement of sialic acids on the cell surface is due to de novo synthesis of glycoproteins and that only a small number of glycoproteins recycle through a trans-Golgi compartment.
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PMID:Intracellular trafficking of cell surface sialoglycoconjugates. 318 95


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