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)

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.
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PMID:The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response. 314 77

A UDP-Gal:Gal beta 1----4GlcNAc-R alpha 1----3- and a UDP-Gal:GlcNAc-R beta 1----4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1----3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1----4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1----3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1----3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1----4GlcNAc-R and Gal beta 1----3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1----3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1----4GlcNAc beta 1----unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3Man beta 1----4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1----3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase from bovine colostrum. In addition alpha 1----3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1----3-galactosyltransferase.
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PMID:Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus. 393 35

In regenerating rat liver the activities of CMP-N-acetylneuraminate hydrolase and UDP-galactose pyrophosphatase were decreased to 40-50% of control values within 35 h after partial hepatectomy. In the same time period the activities of sialyltransferase and galactosyltransferase were increased, and the initial sharp decrease in the carbohydrate content of liver and serum glycoproteins was largely restored. The antiparallel nature of these events is suggestive of an involvement of nucleotide-sugar-hydrolysis enzymes in rat liver glycoprotein biosynthesis.
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PMID:Increased glycosylation capacity in regenerating rat liver is paralleled by decreased activities of CMP-N-acetylneuraminate hydrolase and UDP-galactose pyrophosphatase. 631 60

Golgi vesicle membranes from the Lec2 CHO glycosylation mutant translocate CMP-sialic acid at only 2% the rate of vesicles from wild-type CHO cells. The deficiency is specific, because vesicles from Lec2 cells can translocate UDP-N-acetylglucosamine, adenosine 3'-phosphate 5'-phosphosulfate, and UDP-galactose at rates comparable to those of vesicles from wild-type cells. Complementation analyses show that Lec2 mutants belong to the same genetic complementation group as clone 1021, a CHO mutant of similar phenotype. Both mutants have previously been shown to have a 90% reduction in the sialylation of glycoproteins and gangliosides compared with wild-type cells. However, 1021 cells appear to have normal levels of CMP-sialic acid, sialyltransferase activity, and endogenous acceptors for sialylation. It seems likely that the primary defect in Lec2 and 1021 cells is their inability to translocate CMP-sialic acid across Golgi vesicle membranes.
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PMID:Translocation across Golgi vesicle membranes: a CHO glycosylation mutant deficient in CMP-sialic acid transport. 649 37

The specific activities of UDP-galactose : GM2 galactosyltransferase and CMP-NAN : GM1 sialyltransferase were measured in total particulate fractions of individual hepatocellular carcinomas (hepatomas) and hepatic nodules, induced in the rat by a carcinogenic diet of 0.05% N-2-fluorenylacetamide alternating with a low protein basal diet. In all tumors except the smallest nodules, galactosyltransferase specific activities were increased compared to those of control livers from rats fed only basal diet. When relative specific activities were related to wet weight of nodules larger than 0.1 g were in two populations. Specific activities of nodules in one population overlapped those of poorly differentiated hepatomas whereas specific activities of the second population those of well differentiated hepatomas. Specific activities of the sialyltransferase were also increased above those in control livers but not with the same frequency or to the same extent as for the galactosyltransferase. As with galactosyltransferase, nodules were found in two major populations when specific activities were related to nodule wet weight. The data suggest that increased specific activities of galactosyltransferase and sialyltransferase in hepatic nodules may provide an early phenotypic indicator of diverging differentiation in hepatocarcinogenesis.
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PMID:Gangliosides of liver tumors induced by N-2 fluorenylacetamide III. Galactosyl and sialyl transferases in single carcinomas and nodules. 677 33

Somatic mutations and drugs that either reduce beta 1-6GlcNAc-branching of N-linked oligosaccharides or block the addition of terminal sequences containing galactose and sialic acid have been shown to inhibit tumour growth and metastasis. In an attempt to further define the oligosaccharide sequences that contribute to the malignant phenotype, we have selected spontaneous wheat germ agglutinin-resistant (WGAR) mutants from highly metastatic murine lymphoid tumour cells and characterized four mutant phenotypes. Mutants were selected from VM4, a clone of the MDAY-D2 tumour cell line which had been transfected with the bacterial beta-galactosidase gene (LacZ). VM4 cells retained the malignant phenotype of MDAY-D2 and the cells expressed LacZ, which facilitated the counting of metastases as the tumour cells stained blue when incubated with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal). The most frequently isolated mutant was defective in the transport of UDP-Gal into the Golgi, and as previously observed for this mutation, the cells were non-metastatic and produced very slow-growing solid tumours. Mutants expressing CMP-SA hydroxylase, and consequently glycoconjugates with N-glycolylneuraminic acid (NeuNGc), remained highly metastatic, but grew more slowly than VM4 cells as s.c. tumours in mice. A novel WGAR mutant showing a large increase in Gal beta 1-4GlcNAc:alpha 2-6 sialyltransferase (SA-T) mRNA levels (ST6N) and enzyme activity was observed to be less metastatic and also grew more slowly at the s.c. site of inoculation. Finally, a fourth phenotypic class of WGAR mutants showed a complex phenotype including expression of a beta Gal-binding cell surface lectin and reduced sialylation of glycoconjugates. These results suggest that changes in either the amount, the type or linkage of sialic acid in tumour cell glycoconjugates can affect tumour growth and metastasis.
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PMID:Sialylation and malignant potential in tumour cell glycosylation mutants. 788 Nov 81

Linear and branched glycopeptides containing multiple sialyl-N-acetyllactosamine side chains have been synthesized using a combined chemical and enzymatic approach. Peptide backbones in which beta-GlcNAc-Asn residues were incorporated were obtained in good yields by optimized solid-phase synthesis following the Boc strategy. The resulting multivalent glycopeptides were galactosylated in near-quantitative yields using bovine galactosyltransferase, UDP-galactose, and calf alkaline phosphatase that destroys the inhibiting side product UDP. Subsequent enzymatic sialylation yielded the desired glycopeptides containing asparagine-linked sialyl-N-acetyllactosamine side chains. The compounds were characterized by 1H NMR and FABMS. Recombinant sialyltransferase and CMP-sialate synthetase were used for the enzymatic synthesis of sialosides on a preparative scale. The synthetic glycopeptides were tested as inhibitors of influenza virus to cells, revealing that most of the multivalent sialoglycopeptides exhibit increased binding that depends on the spacing when compared to monovalent compounds. A possible mechanism for increased binding is proposed.
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PMID:Chemical and enzymatic synthesis of multivalent sialoglycopeptides. 814 76

During short incubations of a Golgi apparatus-enriched subcellular fraction from rat liver with UDP-[3H]GlcNAc, label is efficiently transferred to endogenous acceptors. Most of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that it is mainly associated with N-linked oligosaccharides. The glycoprotein acceptors are resistant to proteases unless detergent is added in amounts greater than the critical micellar concentration. This shows that the acceptors are within the lumen of intact compartments, which have the correct topological orientation expected for the Golgi apparatus in intact cells. Structural characterization of the radiolabeled N-linked oligosaccharides shows a variety of distinct neutral and anionic species. The neutral chains include bi-, tri-, and tetra-antennary molecules with terminal beta-[3H] GlcNAc residues. In vitro sialylation shows that some of the tetra-antennary chains have beta 1,3-linked Gal residues on their unlabeled antennae. An unknown modification appears to block the action of beta-galactosidase on these galactosylated oligosaccharides. Chasing the labeling reaction with a mixtures of UDP-Gal, CMP-Neu5Ac, and adenosine 3'-phosphate,5'-phosphosulfate causes an increase in the percent of radiolabeled anionic oligosaccharides. Most of the negative charge is due to sialic acid (Sia), and some appears to be in phosphodiester-linked [3H]GlcNAc. The sialylated oligosaccharides are a mixture of bi-, tri-, and tetra-antennary species with 1-3-Sia residues, and some of the [3H]GlcNAc residues are directly covered with unlabeled Gal and Sia residues. This in vitro approach should recapitulate reactions that occur in the biosynthesis of N-linked oligosaccharides in the Golgi apparatus of the intact cell. Since the conditions during labeling do not permit inter-compartmental transport, the oligosaccharides produced should represent the biosynthetic capabilities of individual Golgi compartments. Evidence is presented for a functional association of GlcNAc transferases I, II, and alpha-mannosidase II, with separation from GlcNAc transferase IV and/or V. The structures also indicate co-compartmentalization of several GlcNAc transferase(s) with beta-galactosyltransferase(s) and sialyltransferase(s). The compartmental organization of the Golgi apparatus is discussed in light of these findings.
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PMID:Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]N-acetylglucosamine. 834 99

The study was designed to understand the effect of orotic acid (OA) on the expression of beta 1,4-galactosyltransferase (GalTase), an enzyme involved in the transfer of galactose from UDP-galactose to a non-reducing terminal N-acetylglucosamine of a glycoprotein or glycolipid. Rats were fed a semisynthetic diet containing 1% OA for 2 weeks and the livers were stimulated to regenerate by two-thirds partial hepatectomy (PH). The level of activity of the enzyme and the steady-state level of hepatic mRNA transcripts of GalTase were determined prior to PH and at 12, 24, 48, 72 h and 10 days after the surgery. The data show that the hepatic activity of GalTase is unaltered in both the control and OA-fed groups until 12 h following surgery, but begins to increase after this time period. In the control group a progressive increase was seen throughout the experimental period following PH. On the other hand in the OA-fed group 24 h after PH the initial increase seen up to 24 h was arrested later on and the activity remained inhibited throughout the rest of the experimental period. The supplementation of 1% OA diet with 0.3% adenine, which is known to reverse the OA-induced imbalance in the nucleotide pool sizes, relieved the inhibition of GalTase activity. The steady-state level of hepatic mRNA paralleled the activity of GalTase at all the time points studied during liver regeneration. The reduction in the level of mRNA transcripts of GalTase in the OA-fed group may not be due to either a general inhibition of synthesis and/or degradation of mRNAs as revealed by a comparison of the expression of beta-galactoside 2,6-sialyltransferase in both the control and OA-fed groups. The study thus suggests an imbalance in nucleotide pools, such as the one induced by OA, may play a role in the regulation of glycosylation by modulating the glycosyltransferases.
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PMID:Effect of orotic acid on beta 1,4-galactosyltransferase during liver regeneration. 850 94

The effects of nucleotides, nucleotide sugars and nucleotide dialdehydes on the activity and kinetics of cytidine 5'-monophospho-N-acetylneuraminic acid:lactosylceramide (alpha 2-->3) sialyltransferase (SAT-1) in microsomes derived from embryonic chick brain were investigated. Although under physiological conditions this enzyme utilizes a CMP-sugar as substrate, it was found that UDP-dialdehyde was an effective inhibitor of SAT-1 activity. CMP-dialdehyde was only slightly more efficient at inhibiting SAT-1 activity. Similar findings were found for the inhibitory effects of UDP versus CMP. In addition, two UDP-sugars (UDP-Gal and UDP-GalNAc) were also slightly inhibitory. Kinetic analyses demonstrate that both UDP- and CMP-dialdehydes are competitive inhibitors of SAT-1 activity. The data suggests that the substrate specificity of microsomal SAT-1 resides more in the sugar moiety, rather than in the nucleotide portion of the substrate.
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PMID:Inhibition of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase by nucleotides, nucleotide sugars and nucleotide dialdehydes. 851 59


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