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)

Incubation of rat jejunal slices in Krebs-Ringer bicarbonate buffer (KRB) required the presence of heat-inactivated horse serum (HHS) in order to show time-dependent release of sialyltransferase into the medium. Sialyltransferase activity could not be detected in the medium when KRB alone or KRB supplemented with either albumin or glycerol was used in the incubations. The viability of the jejunal slices for up to 4 h of incubation was determined by studying the incorporation of glucosamine and leucine into acid-insoluble proteins. Supplementation of KRB with HHS had no beneficial effect on the rate of incorporation of leucine and glucosamine into proteins. KRB medium obtained after different periods of incubation contained higher trypsin-like activity than KRB medium containing HHS. Various antiproteases present as supplements to KRB resulted in the release of sialyltransferase activity from the jejunal slices. Among these antiproteases, alpha 1-proteinase inhibitor (alpha 1-PI) was the most effective. Also, HHS added to KRB immediately following incubation resulted in partial restoration of sialyltransferase activity in the medium, suggesting the presence of anti-proteolytic factors in HHS. The addition of increasing concentrations of heparin to incubations containing HHS caused a decrease in the medium sialyltransferase activity. The heparin-binding fraction (HBF) from HHS, when added to incubations, was able to protect the sialyltransferase released into medium. However, HHS depleted of its heparin-binding fraction by heparin-agarose affinity chromatography was unable to protect the sialyltransferase. HBF was separated into high- and low-molecular-mass fractions (fractions A and B respectively) by gel-filtration chromatography. The capacity to protect the released sialyltransferase was contained in fraction B. Fraction A contained multiple bands on SDS/PAGE and did not protect the enzyme. Fraction B contained a major protein band on the gel which corresponded to the migration of a similar band in human alpha 1-PI. HBF as well as fraction B isolated from HHS showed anti-trypsin-like activity. The results presented indicate that HHS contains a heparin-binding protein(s) similar to human alpha 1-PI which plays a role in the protection of sialyltransferase released from jejunal slices.
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PMID:Heparin-binding serum protein(s) is required for the protection of sialyltransferase released during the incubation of rat jejunal slices. 176 33

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

The presence in the 100,000 g supernatant of rat brain homogenate of an inhibitor of the sialyltransferase has been confirmed. It is also present in chicken and bovine brain and in other rat and bovine organs. The inhibitor has been purified, a preparation with a specific activity 130-fold higher than that of the original 100,000 g supernatant of brain being obtained. It runs as a single peak in polyacrylamide-gel electrophoresis; when run in the presence of SDS, two components appeared. The apparent Mr of the components were 14,800 and 22,400. The inhibitor has been characterized as a heat-stable protein of acidic nature. It has effect on the glycolipid and the glycoprotein sialyltransferase activities but has no effect on the galactosaminyltransferase activity.
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PMID:Purification and characterization of an endogenous inhibitor of the sialyltransferase CMP-N-acetylneuraminate: lactosylceramide alpha 2,6-N-acetylneuraminyltransferase (EC 2.4.99.-). 246 92

We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti-peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD-MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR-containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.
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PMID:Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors. 254 3

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

A stable, sialyltransferase-deficient mutant of Neisseria gonorrhoeae strain F62 totally defective in CMP-NANA-dependent lipopolysaccharide (LPS) sialylation was isolated by insertion mutagenesis with transposon Tn 1545-delta 3 and screened for unlabelled colonies following incubation with CMP-14C-NANA. In contrast to the parental strain which became serum resistant on incubation with CMP-NANA or blood cell extracts, the mutant, JB1, remained serum sensitive. French press extracts of strain F62 catalysed LPS sialylation, but corresponding extracts of the mutant were inactive. Five LPS components were detected by SDS-PAGE in the parental strain. Five components of the same Mr were also found in the mutant. Three identical components were detected by Western blotting using MAb 3F11, which recognizes the Gal beta 1-4GlcNAc groups in the conserved LPS components of F62 which can be sialylated. The mutant, JB1, is therefore deficient in the sialyltransferase that is essential for both LPS sialylation and conversion of serum-sensitive gonococci to serum resistance by either CMP-NANA or blood cell extracts. No evidence was obtained for an LPS sialylation pathway by blood cell extracts that is independent of CMP-NANA.
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PMID:A serum-sensitive, sialyltransferase-deficient mutant of Neisseria gonorrhoeae defective in conversion to serum resistance by CMP-NANA or blood cell extracts. 756 13

CMP-N-acetylneuraminic acid:lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) was purified to homogeneity from a Triton CF-54 extract of young rat brain. The enzyme was separated by affinity chromatography on CDP-Sepharose column and resolved by linear NaCl gradient elution from the same adsorbent. Final purification of GM3-synthase was achieved by chromatography on a "lactosylceramide acid"-Sepharose column and specific elution with lactosylceramide. The enzyme activity was highest at pH 6.5 and required the presence of Triton CF-54 (0.15%) and Mn2+ (10 mM) for its full activity. The product of the reaction catalyzed by the enzyme was identified as GM3 based on its mobility on thin layer chromatographic plates using two different solvent systems. Comparison with several glycolipid substrates showed high specificity of GM3-synthase for lactosylceramide. The apparent Km value for lactosylceramide and CMP-N-acetylneuraminic acid were 80 and 210 microM, respectively. The apparent molecular mass of the enzyme determined on SDS-polyacrylamide gel electrophoresis was 76 kDa.
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PMID:Purification and characterization of CMP-N-acetylneuraminic acid:lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) from rat brain. 825 49

Cell surface carbohydrates have been shown to be altered during cellular differentiation. Alveolar type II (ATII) cells in culture gradually lose their differentiated phenotype. Therefore, the aim of this study was: (1) to characterize changes in terminal carbohydrates of cell surface glycoproteins of rat ATII cells cultured for 1 to 5 days on plastic, and (2) to assess the concomitant changes in sialidase and sialyltransferase activity of ATII cell homogenates. Cells were surface-labeled with potassium-[3H]-borohydride after oxidation by sodium periodate at millimolar concentrations, galactose oxidase or neuraminidase plus galactose oxidase, allowing for the specific labeling of terminal sialic acids, terminal galactose/N-acetylgalactosamine (Gal/GalNAc), or terminal an penultimate Gal/GalNAc residues, respectively. Glycoproteins were separated by SDS-PAGE. On day 1, cells were heavily coated with sialic acids, since no labeling could be introduced with galactose oxidase alone. From day 1 to day 5, we observed a selective and progressive desialylation of two glycoproteins (200 and 165 kD). At the same time, the ATII cells' sialidase activity (pH 4.2) exhibited an 8-fold increase (60.3 +/- 4.0 pmol/min/mg protein on day 1 versus 406.9 +/- 3.7 pmol/min/mg protein on day 5), whereas the sialyltransferase activity increased 2-fold (212 +/- 8 fmol/min/mg protein on day 1 versus 395 +/- 82 fmol/min/mg protein on day 5) and the supernatant sialidase activity was unchanged (2.8 +/- 0.7 pmol/min/ml on day 5). Thus, the phenotypic changes of ATII cells in primary culture are accompanied by a partial cell surface desialylation and an increase in intracellular sialidase activity.
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PMID:Cell surface carbohydrates of rat alveolar type II cells in primary culture. 842 6

Spermatozoa acquire fertilizing ability during passage through the epididymis. Modification of oligosaccharide moieties on sperm surface glycoproteins are some of the biochemical changes believed to be important in the production of functionally mature spermatozoa during passage through the epididymis. In an attempt to understand the mechanism underlying these modifications, we quantified four glycosyltransferase activities (the enzymes that catalyze the transfer of sugar residues from nucleotide sugar donor to the sugar chains on glycoproteins and glycolipids) of spermatozoa and fluid from various regions of the epididymis. Our results are as follows. (1) Only 10-20% of the total glycosyltransferase activities (sialyltransferase, fucosyltransferase, galactosyltransferase, and N-acetyl glucosaminyltransferase) sedimented with the spermatozoa; the remaining 80-90% of the four enzymes were present in soluble form in the epididymal fluid. (2) When the four transferase activities were expressed per 10(6) spermatozoa, only sialyltransferase and fucosyltransferase activities showed maturation-dependent changes. The former enzyme was significantly higher on the proximal caput spermatozoa and the latter on the distal caput spermatozoa. The higher levels of the two enzymes on caput spermatozoa could be due to their binding to the endogenous sugar acceptor molecules on the sperm surface, and subsequent release following sequential sialylation and fucosylation of the molecules in the proximal and distal caput spermatozoa, respectively. (3) When spermatozoa from the proximal and distal caput, corpus, and proximal and distal cauda were incubated with fucose-labeled nucleotide sugar (GDP[14C]fucose), higher levels of radioactivity were routinely incorporated into the spermatozoa from the distal caput. (4) The [14C]fucose-labeled spermatozoa or sperm plasma membranes, when solubilized, resolved on SDS-PAGE, and visualized by autoradiography, showed that the radioactivity had been incorporated into an endogenous acceptor of 86 kDa (major component) and several minor components. Treatment of the solubilized spermatozoa with N-glycanase suggested that the [14C]fucose is mainly present on N-linked oligosaccharide units. These studies demonstrate that some of the sperm surface components are fucosylated during sperm maturation. The potential significance of the in vitro fucosylation of sperm surface components in the production of functionally mature spermatozoa is discussed.
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PMID:Glycosylation of rat sperm plasma membrane during epididymal maturation. 843 31


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