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)

The mechanism of release of Gal beta 1-4GlcNAc alpha-2,6-sialyltransferase (CMP-N-acetylneuraminate: beta-galactoside alpha-2,6-sialytransferase, EC 2.4.99.1) from rat liver during the acute-phase response is due to the action of a cathepsin D-like proteinase that cleaves the trans-Golgi membrane-bound enzyme from a membrane anchor; this allows a major portion of the enzyme containing the catalytic site to escape into the extracellular space [Lammers & Jamieson (1988) Biochem. J. 256, 623-631]. The release of sialytransferase was most effective at pH 5.6, suggesting that release of sialyltransferase from the Golgi in whole cells is dependent on maintaining an acidic environment in the trans-Golgi compartment of the hepatocyte. Golgi membranes contain a proton pump that maintains the acidic pH in these compartments [Glickman, Croen, Kelly & Al-Awquati (1983) J. Cell Biol. 97, 1303-1308; Yamashiro, Tycko & Maxfield (1984) Cell (Cambridge, Mass.) 37, 789-800; Zhang & Schneider (1983) Biochem. Biophys. Res. Commun. 114, 620-625; Anderson & Pathak (1985) Cell (Cambridge, Mass.) 40, 635-643]. Lysosomotropic agents, such as NH4Cl, chloroquine and methylamine can penetrate acidic compartments of the cell, such as the Golgi complex, raise the pH, and thus affect proteolytic cleavage events. The present paper describes the effect of lysosomotropic agents on the release of sialyltransferase from the hepatocyte using liver slices as a whole-cell system. Slices were prepared from control rats and rats suffering from the acute-phase response, where release of sialyltransferase is increased substantially [Lammers & Jamieson (1988) Biochem. J. 256, 623-631; Kaplan, Woloski, Hellman & Jamieson (1983) J. Biol. Chem. 258, 11505-11509]. Release of sialyltransferase was almost abolished in presence of 50 mM-NH4Cl, 50 mM-methylamine or 1 mM-chloroquine. Inhibition of release of sialyltransferase was reversed when the lysosomotropic agents were removed from the medium, showing that these agents are not cytotoxic to the cells under the conditions used. The secretion of rat alpha 1-acid glycoprotein, which is not subject to proteolytic processing in the Golgi complex, was not found to be substantially affected by the presence of lysosomotropic agents. The results suggest that proteolytic cleavage of the catalytic site of sialyltransferase is a process that is significantly affected by the intra-Golgi pH.
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PMID:Studies on the effect of lysosomotropic agents on the release of Gal beta 1-4GlcNAc alpha-2,6-sialytransferase from rat liver slices during the acute-phase response. 250 60

The beta-galactoside alpha-2,6-sialyltransferase represents a member of a family of sialyltransferases which catalyze the terminal addition of sialic acid to maturing carbohydrate chains. We surveyed rat tissues using cDNA probes complementary to coding and noncoding domains of the rat liver alpha-2,6-sialyltransferase. In addition to the expected differences in the level of sialyltransferase mRNA among the tissues, there were dramatic qualitative differences as well. Hepatic sialyltransferase probes hybridize to mRNAs of varying size on Northern blots. A tissue-dependent pattern of expression of these transcripts is documented. Evidence is presented that the multiple transcripts are generated from a common gene sequence. At least one instance of alternate splicing in the generation of the kidney sialyltransferase transcripts is predicted by S1 nuclease analysis. We report the isolation of a rat kidney cDNA clone, RKA, that substantiates this tissue-specific alternate splicing event. The RKA insert, although less than full-length, apparently encodes a polypeptide divergent from the reported hepatic alpha-2,6-sialyltransferase (1). RNA blot analysis indicates that the RKA-type transcripts represent a significant proportion of sialyltransferase RNA in rat kidney. Another class of kidney cDNA clones, RKE, is colinear with the hepatic sialyltransferase sequence. RNA blots probed for the divergent and common regions suggest that complex processing pathways are operative in the tissue-specific expression of sialyltransferase mRNA.
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PMID:Tissue-specific expression of beta-galactoside alpha-2,6-sialyltransferase. Transcript heterogeneity predicts a divergent polypeptide. 279 63

This report describes the primary structure of a rat liver beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1), a Golgi apparatus enzyme involved in the terminal sialylation of N-linked carbohydrate groups of glycoproteins. The complete amino acid sequence was deduced from the nucleotide sequence of cDNA clones of the enzyme. The primary structure suggests that the topology of the enzyme in the Golgi apparatus consists of a short NH2-terminal cytoplasmic domain, a 17-residue hydrophobic sequence which serves as the membrane anchor and signal sequence, and a large lumenal, catalytic domain. NH2-terminal sequence analysis of a truncated form of the enzyme, obtained by purification from tissue homogenates, reveals that it is missing a 63-residue NH2-terminal peptide which includes the membrane binding domain. These and supporting results show that soluble forms of the sialyltransferase can be generated by proteolytic cleavage between the NH2-terminal signal-anchor and the catalytic domain.
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PMID:Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor. 312 4

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

Rat liver beta-galactoside alpha-2,6-sialyltransferase and Vibrio cholerae sialidase were used, in conjunction with CMP-N-acetyl-[3H]neuraminic acid, to probe the glycoconjugate distribution, sialylation state, and level of penultimate Gal beta 1-4GlcNAc residues on the surfaces of murine thymic lymphocytes. We report a detailed characterization of this sialyltransferase-mediated labeling system. Exogenous sialylation of intact cells is dependent on transferase, sugar nucleotide donor, cell number, and incubation time. Additionally, we have demonstrated that the system labeling the cell surface is noncytotoxic and nonmetabolic and is interacting with the entire cell population. Analysis of the exosialylated structures indicates that the sialyltransferase specifically produces an alpha 2-6 linkage on N-linked oligosaccharides. Using this labeling system, we have probed the cell surface saccharide structures of murine thymocytes and demonstrated that most Gal beta 1-4GlcNAc residues are sialylated in the native state. However, one antigen, T200 (Ly-5), is strikingly undersialylated when compared to other cell surface glycoproteins (e.g., Thy 1.2). Upon analysis of exogenously sialylated oligosaccharides, labeled sialic acid was found almost exclusively on monosialylated structures with the remainder on bisialylated oligosaccharides. This suggests that the purified sialyltransferase is very precise in its recognition of oligosaccharides present on the surface of living thymic lymphocytes. This paper illustrates the combined uses of specific glycosidases and glycosyltransferases and how they can be employed in the detailed study of selected cell surface saccharide structures on living nucleated cells.
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PMID:Sialyltransferases as specific cell surface probes of terminal and penultimate saccharide structures on living cells. 330 6

Elevated levels of glycoprotein:sialyltransferase activity (EC 2.4.99.1; CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetylneuraminyltransferase) were found in human malignant neoplastic tissues compared to normal, benign, and "preneoplastic" tissues. This increase was not due to the cell density of the tissue. Elevated levels of certain proteases and glycosidases were also found. The increase in transferase activity may be associated with altered membrane synthesis in the neoplastic state; changes in the activity of degradative enzymes may be associated with tumor invasiveness and maintenance of the neoplastic state. Measurements on human tumors are possibly more directly relevant to cancer than those described for transformed fibroblastic cells in vitro.
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PMID:Enzyme activity in invasive tumors of human breast and colon. 436 73

GMP-N-Acetylneuraminate: galactosyl-glycoprotein sialytransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity was identified in the human cervical epithelium. The enzyme has a pH optimum of 6.0, a temperature optimum of 28 degrees C, and demonstrates a partial requirement for Triton X-100. Michaelis constants for asialofetuin and CMP-N-acetyl[14C]neuraminic acid are 0.64 . 10(-5) M (expressed as the concentration of terminal galactose residues) and 2.05 . 10(-5) M, respectively. Sialytransferase demonstrated minimal affinity for the low molecular weight acceptors tested, and may have a requirement for a glycoprotein acceptor having a terminal N-acetyllactosamine (Gal beta (1 leads to 4)GlcNAc) type structure. Cytidine nucleotides are potent inhibitors of the sialyltransferase reaction; CMP acts as a competitive inhibitor.
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PMID:Glycosyltransferases of the human cervical epithelium. II. Characterization of a CMP-N-acetylneuraminate: galactosyl-glycoprotein sialyltransferase. 616 92

The distribution of multiple forms of galactosyltransferase (EC 2.4.1.22) and sialyltransferase (EC 2.4.99.1) from the microsomes and Golgi complex membrane fractions of rat liver was investigated. Three fractions of Golgi membranes, namely GF1, GF2, and GF3, differing in their morphology and marker enzyme activity, were obtained. A simultaneous increase of glycosyltransferases under study was observed in fractions GF3 less than GF2 less than GF1. Using isoelectrofocusing, the presence of at least 6-8 forms of galactosyl- and sialyltransferases in the microsomes and Golgi fraction was revealed. The distribution patterns of multiple forms along the pH gradient for each membrane fraction were found to be identical. However, the ratios of highly active and low active forms were specific for each fraction. The similarity of multiple form spectra for galactosyl-and sialyltransferase suggest their tight functional interaction and a possible "en block" packing of membrane glycosyltransferases.
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PMID:[Multiple forms of glycosyltransferases in Golgi complex membrane fractions]. 641 72

In order to study structure-function relationships of lysosomal enzymes, human liver beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) has been purified by an extraction/affinity chromatography/ion-exchange procedure. The isoenzymes A and B, native as well as neuraminidase-treated, were incubated with a partially purified preparation of bovine colostrum sialyltransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1). Native beta-N-acetylhexosaminidases were found to be poor acceptors for the sialyltransferase used. However, incorporation of sialic acid into neuraminidase-treated beta-N-acetylhexosaminidase A and B amounted to a 58 to 72% saturation of the theoretical acceptor sites, respectively. The acceptor specificity of the sialyltransferase suggests that Gal beta(1 leads to 4)-GlcNAc units may be present on at least part of the beta-N-acetylhexosaminidase A and B molecules. However, oligomannosidic-type chains may also occur on the lysosomal enzyme, as shown by sugar composition of the enzyme. The presence and/or amount of sialic acid residues does not appear to affect the kinetic properties of beta-N-acetylhexosaminidase A and B towards 4-methylumbelliferyl glycoside substrate.
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PMID:Sialylation in vitro of purified human liver beta-D-N-acetylhexosaminidase. 645 69

L-Fucose and N-acetylneuraminic (sialic) acid occupy terminal positions on the oligosaccharide side-chains of human cervical mucin but the addition of both these monosaccharides to the same carbohydrate acceptor residue is kinetically unfavourable. The following evidence suggests that the levels of L-fucose are more sensitive to regulation than those of N-acetylneuraminic acid: (1) tissue levels of sialyltransferase (EC 2.4.99.1) activity are 20-30 times greater than those of fucosyltransferase (EC 2.4.1.68); (2) both glycosyltransferases are susceptible to inhibition by their nucleotide products but a comparison of the Ki and the apparent Km of these enzymes shows that modulation of fucosyltransferase is more probable; (3) Postsecretory removal of L-fucose from cervical mucin is probably due to the high levels of mucus-associated alpha-L-fucosidase. Furthermore the activity of this enzyme is probably modulated by the pH gradient within the cervix. Mucin glycosylation can be visualized by autoradiography using [3H]L-fucose applied to cervical explants in organ culture. Mucus production during this process is not sensitive to exogenous ovarian steroid hormones, though in other aspects the secretory process appears normal. It is proposed that the cyclicity of mucus rheology is not directly influenced by an action of these hormones on mucin synthesis or hydration.
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PMID:Terminal glycosylation in human cervical mucin. 656 36


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