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:2.4.99.10 (sialyltransferase)
1,547 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Smooth membrane preparations of 13-day embryonic chicken livers, characterized by electron microscopy and marker enzyme analyses, have been found to contain sialyltransferase activity which displayed precise acceptor specificity. One sialyltransferase transferred N-acetyl-neuraminic acid (NANA) and gal beta 1 leads to 4glycNAc beta 1 leads R structures. Evidence based on competition studies suggests that a second enzyme is present transferring this sugar to a gal beta 1 leads to 3gal-NAc alpha 1 leads to R structure. The enzyme capable of adding NANA to gal beta 1 leads to 4glcNAc beta 1 leads to R structures has a pH optimum of 5.5, a temperature optimum of 30 degrees C, and half-saturating values of 17 muM for CMP-NANA and 180 muM for galactoside termini on desialyzed alpha 1-acid glycoprotein. It is activated about 10-fold by Triton X-100, has no exogenous divalent cation requirement, and is inhibited by CTP, CDP, and CMP. The enzyme requires carbohydrate structures underlying the gal beta 1 leads to 4glcNAc terminus for maximal catalytic activity; the necessity of such precise specificities of sialyltransferases is discussed in the light of recent structural evidence for the carbohydrate moieties of several glycoproteins.
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PMID:The specificity of sialyltransferase activity in smooth membrane fractions of embryonic chicken liver. 722 24

Sialyltransferase activity of the intima-media of arterial walls is located in microsomes. The solubilization of enzyme by Triton X-100, purification by chromatography and isoelectric focusing lead to two isoenzymes (pHi 4.5 and 6.4), the second is widely predominant. Aortic sialyltransferase is sensitive to nucleotides, Mn++ dependent, and the affinity for substrate N-acetyl-neuraminic acid is 300 microM.
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PMID:[Glycoconjugate biosynthesis in the arterial wall. VIII. Separation of molecular types in microsomal sialyltransferase]. 733 43

Biosynthesis and intracellular transport of recombinant human full-length beta 1,4 galactosyltransferase (GT) and full-length alpha 2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C., Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur. J. Biochem. 212, 113-120]. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamine as acceptor substrates could readily be detected. Analysis by 1H-NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold. rGT and rST could be immunoprecipitated from their [35S]Met-labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of beta-galactosidase-GT or beta-galactosidase-ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin-treated [35S]Met-labelled transformed yeast cells or labelling with radio-active sugars both indicated that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi-specific man-alpha 1,6-man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus. These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.
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PMID:Human beta 1,4 galactosyltransferase and alpha 2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum. 814 35

This communication examines the possibility that nitric oxide (NO) production by endothelial cells results from changes in cell membrane fluidity. Lysophosphatidylcholine (LPC) alters fluidity of the endothelial cell membranes causing vascular relaxation. Through membrane alterations LPC influences function of a number of membrane receptors and modulates enzyme activity. As a result of detergent action, lysophosphatidylcholine (LPC) causes activation of guanylate cyclase, stimulates sialyltransferase and regulates protein kinase C activity. It has already been demonstrated that ionic detergents, such as Triton X-100 also cause vascular relaxation, possibly induced by NO production from endothelial cells. It is postulated that production of nitric oxide results from changes in membrane viscosity; this may represent a mechanism for its regulation in biological systems.
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PMID:Membrane function and vascular reactivity. 839 7

In vivo, gonococci encounter a myriad of conditions not present in vitro. At some stages of infection and disease, gonococci may grow anaerobically, probably by using sodium nitrite as a terminal electron acceptor. Also, gonococci sialylate their lipooligosaccharide (LOS) in vivo, by using low concentrations of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) present in host tissue. This sialylation is responsible for the acquired resistance of gonococci to both normal and immune human serum. Given that gonococci grown in the absence of oxygen or in the presence of CMP-NANA probably more closely resemble gonococci grown inside a human host, we studied the serum resistance of gonococci cultivated under these conditions. In the absence of CMP-NANA, anaerobically grown (anaerobic) gonococci were somewhat less sensitive to serum killing than were aerobically grown (aerobic) gonococci. However, anaerobic gonococci grown with 6 micrograms of CMP-NANA per ml exhibited almost complete serum resistance, while aerobic gonococci required 16-fold-higher CMP-NANA concentrations to achieve significant serum resistance. Anaerobic gonococci incubated in CMP-NANA converted to serum resistance two to three times faster than did similarly treated aerobic gonococci and incorporated up to six times as much sialic acid into their LOS. Gonococci can express several different LOS molecules. Anaerobic gonococci expressed the LOS molecule that acts as an acceptor for sialic acid from CMP-NANA in greater quantity than aerobic gonococci did. Finally, Triton X-100 extracts of anaerobic gonococci contained about four times more sialyltransferase activity than did extracts of aerobic gonococci. Sialyltransferase activity in these extracts was not inhibited by oxygen or enhanced by anaerobiosis. These data indicate that anaerobic conditions lead to altered LOS biosynthesis and to induction of sialyltransferase activity in gonococci. In vivo, where decreased oxygen levels and relevant concentrations of CMP-NANA are found, gonococci could readily become resistant to killing by normal and immune human serum.
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PMID:Anaerobic growth and cytidine 5'-monophospho-N-acetylneuraminic acid act synergistically to induce high-level serum resistance in Neisseria gonorrhoeae. 847 54

A method for the assay of CMP-NeuAc:(NeuAc alpha 2-->8)n (colominic acid) sialyltransferase activity was developed. Using a 1-day-old rat brain membrane fraction as an enzyme preparation optimal activity was obtained at pH 6.5, 0.3% Triton X-100, and 5 mM MnCl2. However, no absolute cation requirement was found as EDTA only partially inhibited the activity. Within a concentration range of 0.3-3 mg colominic acid (which consists of a mixture of oligomers of alpha 2-->8-linked sialic acid) per 50 microliters a V of 0.61 nmol per mg protein h-1 was estimated while a half-maximal reaction velocity was obtained at a concentration of 1.75 mg per 50 microliters. High performance anion-exchange chromatography of the radioactive products formed in the reaction showed that sialic acid oligomers ranging in size from a degree of polymerization (DP) of 2 up to at least DP 9 could serve as acceptor substrates. Comparison of the acceptor properties of DP 3 and DP 6 showed that the larger oligomer was acted upon with a 10-fold higher efficiency. Periodate oxidation of the products followed by reduction and hydrolysis yielded the C7 analogue of NeuAc as the only radioactive product, indicating that under the conditions of the assay only a single sialic acid residue was introduced into the acceptor molecules. Using the assay it appeared that in rat brain the activity of this sialyltransferase decreased six-fold during postnatal development to the adult stage. The assay method was also applied to lysates of several neuroblastoma and small cell lung tumour cell lines, which differ in the expression of polysialic acid as well as of the neural cell adhesion molecule NCAM, a major carrier of this polymer. Activity of the sialyltransferase appeared to be correlated with the expression of polysialic acid present on NCAM. These results indicate that this sialyltransferase might function in the process of poly-sialylation.
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PMID:CMP-NeuAc:(NeuAc alpha 2-->8)n (colominic acid) sialyltransferase activity in rat brain and in tumour cells that express polysialic acid on neural cell adhesion molecules. 874 61

Rat liver Golgi membranes were found to contain an enzyme that can transfer sulphate from 3'-phosphoadenosine 5'-phosphosulphate (PAPS) to C-6 of the terminal GlcNAc in beta-linkage to mannose and has properties indicating that it is involved in the synthesis of the NeuAc alpha 2-3(6)Gal beta 1-4GlcNAc(6-SO4) sequences observed in the N-linked carbohydrate units of various glycoproteins. Assays performed with [35S]PAPS (Km 0.67 microM) and GlcNAc beta 1-6Man alpha 1-O-Me (GnMaMe) acceptor (Km 0.71 mM) indicated that the sulphotransferase had a pH optimum of approx. 7.0 and is markedly stimulated by Mn2+ ions (maximum approx. 15 mM) and Triton X-100 (0.05-0.1%). Hydrazine/nitrous acid/NaBH4 treatment of the 35S-labelled product yielded radiolabelled 2,5-anhydromannitol(6-SO4). The sulphated GnMaMc product of the GlcNAc-6-O-sulphotransferase could be galactosylated by a rat liver Golgi enzyme that was shown to have the same properties as the UDP-Gal:GlcNAc beta-1,4-galactosyltransferase from bovine milk. Competition studies performed with GlcNAc and GlcNAc-6-SO4 furthermore indicated that the same liver enzyme acted on both acceptors to produce Gal beta 1-4GlcNAc and Gal beta 1-4GlcNAc(6-SO4) with Km values of 1.04 and 1.68 mM respectively. Because the sulphated N-acetyl-lactosaminc could in turn serve as an acceptor for rat liver sialyltransferase, it seems that this enzyme, together with the Golgi galactosyltransferase and the GlcNAc-6-O-sulphotransferase, could act in concert in assembling the NeuAc alpha 2-3(6)Gal beta 1-4GlcNAc(6-SO4) branches of complex N-linked oligosaccharides.
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PMID:Characterization of a rat liver Golgi sulphotransferase responsible for the 6-O-sulphation of N-acetylglucosamine residues in beta-linkage to mannose: role in assembly of sialyl-galactosyl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides. 887 Jun 71

Sialic acids from the liver and serum of guinea-pig are composed of N-acetylneuraminic acid (Neu5Ac; 85% and 61%, respectively), N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2; 10% and 32%, respectively) and N-glycolylneuraminic acid (Neu5Gc; 5% and 7%, respectively), besides traces of N-glycolyl-4-O-acetylneuraminic acid in serum. The analysis was carried out using thin-layer chromatography, high-performance liquid chromatography, electron impact ionization mass spectrometry, and different enzymes (sialidase, sialate esterase, and sialate-pyruvate lyase after hydrolysis and purification of the sialic acids by ion-exchange chromatography). We showed that this O-acetylation of sialic acids is due to the activity of an acetyl-coenzyme A:sialate-4-O-acetyltransferase (EC 2.3.1.44), which occurs together with sialyltransferase activity in Golgi-enriched membrane fractions of guinea-pig liver. The enzyme operates optimally at 30 degrees C in 70 mM potassium phosphate buffer at pH 6.7 and in the presence of 90 mM KCI with an apparent KM for AcCoA of 0.6 1microM and a Vmax of 20 pmol/mg protein x min. The enzyme is inhibited by coenzyme A in a mixed-competitive manner (Ki = 4.2 microM), as well as by parachloromercuribenzoate, MnCl2, saponin and Triton X-100.
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PMID:Enzymatic 4-O-acetylation of N-acetylneuraminic acid in guinea-pig liver. 1005 93

The ST6Gal I is a sialyltransferase that functions in the late Golgi to modify the N-linked oligosaccharides of glycoproteins. The ST6Gal I is expressed as two isoforms with a single amino acid difference in their catalytic domains. The STcys isoform is stably retained in the cell and is predominantly found in the Golgi, whereas the STtyr isoform is only transiently localized in the Golgi and is cleaved and secreted from a post-Golgi compartment. These two ST6Gal I isoforms were used to explore the role of the bilayer thickness mechanism and oligomerization in Golgi localization. Analysis of STcys and STtyr proteins with longer transmembrane regions suggested that the bilayer thickness mechanism is not the predominant mechanism used for ST6Gal I Golgi localization. In contrast, the formation and quantity of Triton X-100-insoluble oligomers was correlated with the stable or transient localization of the ST6Gal I isoforms in the Golgi. Nearly 100% of the STcys and only 13% of the STtyr were found as Triton-insoluble oligomers when Golgi membranes of COS-1 cells expressing these proteins were solubilized at pH 6.3, the pH of the late Golgi. In contrast, both proteins were found in the soluble fraction when these membranes were solubilized at pH 8.0. Analysis of other mutants suggested that a conformational change in the catalytic domain rather than increased disulfide bond-based cross-linking is the basis for the increased ability of STcys protein to form oligomers and the stable localization of STcys protein in the Golgi.
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PMID:Formation of insoluble oligomers correlates with ST6Gal I stable localization in the golgi. 1078 4

Alpha-2,3-sialyltransferase (Lst) is expressed on the outer membrane of Neisseria gonorrhoeae and Neisseria meningitidis and sialylates surface lipooligosaccharide (LOS), facilitating resistance to complement-mediated killing. The enzyme is constitutively expressed from a single gene (lst) and does not undergo antigenic or phase variation. We observed that Triton X-100 extracts of N. gonorrhoeae strain F62 contain about fivefold more sialyltransferase (Stase) activity than extracts of N. meningitidis strain MC58 [symbol: see text]3 a serogroup B acapsulate mutant. We confirmed and expanded upon this observation by showing that extracts of 16 random N. gonorrhoeae isolates contain various amounts of Stase activity, but, on average, 2.2-fold-more Stase activity than extracts of 16 N. meningitidis clinical isolates, representing several serogroups and nongroupable strains. Northern and real-time reverse transcription-PCR analysis of lst transcript levels in N. gonorrhoeae and N. meningitidis revealed that N. gonorrhoeae strains express more lst transcript than N. meningitidis strains. Although transcript levels correlate with average Stase activity observed in the two species, there was not a direct correlation between lst transcript levels and Stase activity among individual isolates of each species. Comparison of lst upstream (5'lst) regions of N. gonorrhoeae and N. meningitidis revealed striking sequence differences characteristic of the two pathogens. N. gonorrhoeae 5'lst regions possess 30-bp and 13-bp elements present as single elements or as tandem repeats that exist only as single elements in the 5'lst regions of N. meningitidis isolates. In addition, the 5'lst regions of N. meningitidis strains have 105-bp transposon-like Correia elements which are absent in N. gonorrhoeae. Chromosomal N. gonorrhoeae 5'lst::lacZ translational fusions expressed 4.75 +/- 0.09-fold (n = 4) higher beta-galactosidase (beta-gal) activity than N. meningitidis 5'lst::lacZ fusions in a host-independent manner, indicating differential expression is governed at least in part by sequence variations in the 5'lst regions. Reporter fusion assays and promoter-mapping analysis revealed that N. gonorrhoeae and N. meningitidis use different promoters with different strengths to transcribe lst. In N. gonorrhoeae, a strong sigma 70 promoter 80 bp upstream of the translational start site is used to transcribe lst, whereas this promoter is inactive in N. meningitidis. In N. meningitidis, a weak sigma 70 promoter at the 3' terminus of a 105-bp Correia repeat-enclosed element 99 bp upstream of the translational start site is used to transcribe lst. We conclude that differential Stase expression between N. gonorrhoeae and N. meningitidis is due at least in part to differential lst gene transcription.
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PMID:Differential expression and transcriptional analysis of the alpha-2,3-sialyltransferase gene in pathogenic Neisseria spp. 1662


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