Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.99.10 (sialyltransferase)
 1,547 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Membrane-associated sialyltransferase complexes in Escherichia coli K-235 can be dissociated by lipid deletion and reassembled by the addition of undecaprenyl phosphate, a unique membrane-bound lipid coenzyme. Following disruption of the cells by pressure disintegration and centrifugal fractionation, the sialyltransferase activity is assocatied with both a "particulate" and "soluble" complex. Kinetic studies as well as sugar nucleotide, metal ion, pH, ammonium sulfate, and thiol reagent requirements showed these two complexes contained functionally identical enzymatic activities. Isopycnic sucrose density gradient centrifugation studies carried out on unfractionated total membranes established that these sialytransferase activities were associated with membrane hybrids composed of different relative amounts of inner and outer membranes. Enzyme localization studies employing DPNH oxidase, a marker for the inner membrane, and relative phospholipid to protein composition determinations in the two complexes, provided added support for this conclusion. Sialyl polymer synthesis was not dependent on the incorporation of other monosaccharides and had no demonstrable metal ion requirement. Kinetic studies showed that the Km for cytidine 5-monophospho-N-acetylneuraminic acid in intact soluble and particulate enzyme preparations was 8.1 times 10-5M and 9.2 times 105M, respectively. Similarly, both enzyme complexes had nearly identical Vmax values. Following reassembly of delipidated enzyme preparations, however, there was a 10-fold increase in the Km value for the particulate enzyme and a 3-fold increase for the soluble enzyme. This increase was accompanied by an increase of approximately the same magnitude in the Vmax values. Since the lipid coenzyme was limiting in intact enzyme preparations, the increase in Vmax reflected an increase in the concentration of the active lipid in reconstituted complexes. Sialyl polymer synthesis in intact membrane preparations was stimulated by the exogenous addition of lipid. Insertion of the carrier lipid was dependent on temperature. At 37 degrees, a 120% increase in sialytransferase activity was observed while only a 35% increase was observed at 30 percent. At 20 degrees, no stimulation occurred. Fluidity of the lipid phase is apparently required for proper function of this membraneassociated enzyme complex. Thus, at 20 degrees, a temperature below the membrane lipid transition temperature, the lipids are relatively immobile.
 ...
PMID:Properties of membrane-associated sialyltransferase of Escherichia coli. 109 67

The Golgi complex is composed of at least four distinct compartments, termed the cis-, medial, and trans-Golgi cisternae and the trans-Golgi network (TGN). It has recently been reported that the organization of the Golgi complex is disrupted in cells treated with the fungal metabolite, brefeldin-A. Under these conditions, it was shown that resident enzymes of the cis-, medial, and trans-Golgi return to the ER. We report here that 300-kD mannose 6-phosphate receptors, when pulse-labeled within the ER of brefeldin-A-treated cells, acquired numerous N-linked galactose residues with a half time of approximately 2 h, as measured by their ability to bind to RCA-I lectin affinity columns. In contrast, Limax flavus lectin chromatography revealed that less than 10% of these receptors acquired sialic acid after 8 h in brefeldin-A. Two lines of evidence suggested that proteins within and beyond the TGN did not return to the ER in the presence of brefeldin-A. First, the majority of 300-kD mannose 6-phosphate receptors present in the TGN and endosomes did not return to the ER after up to 6 h in brefeldin-A, as determined by their failure to contact galactosyltransferase that had relocated there. Moreover, although mannose 6-phosphate receptors did not acquire sialic acid when present in the ER of brefeldin-A-treated cells, they were readily sialylated when labeled at the cell surface and transported to the TGN. These experiments indicate that galactosyltransferase, a trans-Golgi enzyme, returns to the endoplasmic reticulum in the presence of brefeldin-A, while the bulk of sialyltransferase, a resident of the TGN, does not. Our findings support the proposal that the TGN is a distinct, fourth compartment of the Golgi apparatus that is insensitive to brefeldin-A.
 ...
PMID:Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. 216 98

1. Subcellular fractions isolated from livers of 19-day-old chicken embryos were analyzed in order to assess whether liver mitochondria contained glycosylated proteins or had mannosyl- or sialyl-transferases that could transfer sugars to mitochondrial macromolecules. 2. Proteins in liver mitochondrial membranes and matrix fractions were screened for their affinities for concanavalin A (Con A). 3. After separation by gel electrophoresis under denaturing conditions, a significant number of the proteins bound [125I]Con A, and the binding of the lectin was substantially inhibited by alpha-methyl-D-mannoside. 4. In addition, radio-iodinated matrix proteins were screened for lectin-binding properties by chromatography on Con A covalently linked to agarose. 5. A number of proteins, representing 14% of those loaded onto the column, became tightly bound to the agarose-linked lectin, and the molecular weights of several of those proteins are reported. 6. Mannosyltransferase activities were measured in fractions highly enriched for mitochondria. 7. In the reactions, mannose was transferred from guanosine diphosphomannose to materials insoluble in 0.3% trichloroacetic acid or in chloroform:methanol (2:1). 8. The fractions also catalyzed the transfer of mannose to materials extractable in chloroform:methanol and which migrated with the Rf of dolichol phosphate on Silica Gel H. 9. Dolichol phosphate stimulated the transfer of mannose to those materials extractable in the organic solvents. 10. Marker enzyme analyses indicated that the mannosyl transferase activity in the mitochondrial fraction could not be accounted for entirely by contaminating microsomal membranes. 11. Although sialyltransferase activity was detected also in the mitochondrial fractions, the levels of the activity and the kinetics of the reactions indicated that Golgi membranes were most likely the sources of the enzyme.
 ...
PMID:Mitochondrial biogenesis: do liver mitochondria contain glycoproteins and glycosyltransferases? 228 16

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.
 ...
PMID:Effect of desipramine on a glycoprotein sialyltransferase activity in C6 cultured glioma cells. 229 42

We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells.
 ...
PMID:Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus. 296 50

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.
 ...
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

Neisseria meningitidis serogroup B strain M986 was examined for the involvement of lipid intermediate(s) participating in the biosynthesis of the sialic acid capsular polysaccharide. The addition of exogenous undecaprenyl phosphate, phosphatidylethanolamine, or phosphatidylglycerol to particulate membranes, in the presence of cytidine 5'-monophosphosialic acid, resulted in the stimulation of sialyltransferase activity specifically by undecaprenyl phosphate. Sialyltransferase activity, after delipidation of particulate membrane proteins, was specifically reconstituted by undecaprenyl phosphate. After the addition of 14C-labeled cytidine 5'-monophosphosialic acid to particulate membranes, the level of labeled lipid intermediate(s), extracted by chloroform-methanol (2:1), increased up to a maximum level between 3.75 and 5.0 min, which subsequently decreased to a lower steady-state level. Pulse-chase experiments revealed a transient, solvent-extractable, lipid-linked component. The extracted N-acetylneuraminic acid was in polymeric form. Sequential oxidation and reduction of the extracted radioactivity followed by neuraminidase treatment revealed an average degree of polymerization of four or five N-acetylneuraminic acid residues. Bacitracin-sensitive peptidoglycan was synthesized in vitro by particulate membranes. Cross-competition experiments between peptidoglycan and capsular polysaccharide synthesis by preincubation of precursors of one pathway during synthesis of the other revealed a competitive effect for a common component. This component was believed to be a common pool of undecaprenyl phosphate. A model for the production and regulation of the capsular polysaccharide is proposed.
 ...
PMID:Role of lipid intermediate(s) in the synthesis of serogroup B Neisseria meningitidis capsular polysaccharide. 391 90

The activities of ten enzymes involved in sialic acid metabolism were measured in colonic mucosal cells from rats and compared with those in liver. A methodology was devised that enabled all ten enzyme activities to be evaluated in a single rat colon preparation. Enzyme assays with radioactively labelled substrates were developed for maximum sensitivity, and the identification of substrates and products was carefully checked to assess the contribution of contaminants to enzyme reactions with low activity. The activities of most enzymes involved in the biosynthesis of N-acetyl-D-neuraminic acid (NeuAc) from UDP-N-acetyl-D-glucosamine were found to be more than 20-fold lower than those in liver. The activities of CMP-NeuAc synthase, N-acetyl-D-glucosamine 2-epimerase, N-acetyl-D-glucosamine kinase, sialyltransferase and sialidase were similar to or 2-4-fold lower than in liver. The biosynthesis of NeuAc via its 9-phosphate was demonstrated in the 100 000 g supernatant of colonic-cell homogenates by enzymic assay and precursor experiments with N-acetyl[14C]-mannosamine. No alternative route for NeuAc formation could be detected. The 100 000g supernatant fractions of liver, kidney and colonic mucosal cells utilized N-acetyl[14C]mannosamine with differing efficiencies. Radioactive products identified as sialic acid biosynthetic intermediates amounted to 49%, 0.04% and 5.6% of added precursor in liver, kidney and colon respectively. Catabolism of labelled precursor to non-hexosamine products was high in kidney and colonic mucosal-cell fractions.
 ...
PMID:The metabolism of sialic acids in isolated rat colonic mucosal cells. 397 62

The postnatal development of skeletal muscle is characterized by changes in membrane function associated with N-linked glycoproteins. In the present study, early reactions involved in the synthesis of the dolichol-linked core oligosaccharide were examined in neonatal and adult rabbit skeletal muscle sarcoplasmic reticulum membranes. The initial rate of N-acetylglucosamine incorporation in the presence of exogenous dolichol phosphate was similar between neonate and adult (3.5-4.1 pmol of GlcNAc/min/mg). The Km values for UDP-GlcNAc and exogenous dolichol phosphate were similar. Tunicamycin (0.04-0.08 micrograms/ml) inhibited N-acetylglucosamine incorporation by 50%. UDP-GlcNAc pyrophosphatase activity was greater in neonatal membranes than adult (840 versus 350 pmol of GlcNAc-1-P/min/mg), explaining, in part, the greater enhancement of neonatal GlcNAc incorporation by pyrophosphatase inhibitors. Nucleotide-sugar pyrophosphatase inhibitors (alpha, beta-methylene ATP and dimercaptopropanol) increased the capacity of neonatal activity 4-fold and adult enzyme 2-fold. Analysis of dolichol-linked products by mild acid hydrolysis however, revealed that neonate had higher capacity for N,N'-diacetylchitobiosyl(pyro)phosphoryldolichol synthesis than adult. Mannosyltransferase and glucosyltransferase were elevated 6- and 5-fold in neonate compared to adult membranes. Neonate exhibited 4-fold greater GDP-Man pyrophosphatase activity than adult (500 versus 125 pmol of Man-1-P/min/mg). The Km for GDP-Man increased in the presence of exogenous dolichol phosphate. Increasing concentrations of exogenous dolichol phosphate did not equalize neonate and adult mannosyltransferase activity, indicating that the decline in activity during development was not due to a decrease in a pool of dolichol phosphate accessible to mannosyltransferase. Glucosyltransferase for the synthesis of glucosylphosphoryldolichol was also elevated 5-fold in neonatal compared to adult sarcoplasmic reticulum (7 versus 1.4 pmol of Glc/min/mg). In a previous study, it was reported that glycoprotein sialyltransferase activity decreased by a factor of 6.5 during the postnatal maturation and that total membrane hexose content of sarcoplasmic reticulum decreased by a factor of 8. Together, these results suggest that the postnatal development of skeletal muscle is characterized by coordinated changes in the expression of enzymes involved in both the "early" and "late" reactions of N-linked oligosaccharide biosynthesis.
 ...
PMID:Formation of dolichol-linked sugar intermediates during the postnatal development of skeletal muscle. 631 23

Escherichia coli K1 synthesizes a polysialic acid capsule when grown at 37 but not 15 degrees C. The derangement in sialyl polymer synthesis appears to result from the inability of 15 degrees C membranes to synthesize or assemble a functional endogenous acceptor (Troy, F.A., and McCloskey, M.A. (1979) J. Biol. Chem. 254, 7377-7387). Membranes from cells grown at 15 degrees C spontaneously gained the ability to synthesize sialyl polymer after incubation at 33 degrees C for 2-4 h. The incubation-dependent activation of the endogenous synthesis of sialyl polymer in 15 degrees C membranes possessed two unusual features. First, the sialyltransferase was localized in a low density vesicle fraction (LDV; rho = 1.11 g/cm3). Second, this fraction catalyzed protein synthesis, and protein synthesis was required for activation. A study of the LDV fraction showed: 1) their light density resulted from a 5- to 8-fold enrichment in lipid phosphate to protein ratio and their sialyltransferase activity was enriched 40-fold compared with unfractionated total membranes; 2) they contained proteins characteristic of inner and outer membranes including leader peptidase and lipoprotein; 3) they constituted 8% of the mass of unfractionated total membranes yet contained all of the endogenous sialyltransferase activity in 15 degrees C membranes. In contrast, LDV from 37 degrees C grown cells accounted for 4.8% of the membrane mass and only 12.5% of the endogenous sialyltransferase activity; 4) they were multilamellar and averaged 0.7 mu in diameter. Based on these results, we believe the LDV fraction is of physiological importance in sialyl polymer synthesis. Growth at 15 degrees C allowed identification and study of the LDV fraction possibly because of the altered thermotropic properties of the membrane phospholipids that occur when E. coli is grown at low temperature.
 ...
PMID:Biosynthesis and assembly of the polysialic acid capsule in Escherichia coli K1. Role of a low-density vesicle fraction in activation of the endogenous synthesis of sialyl polymers. 638 2


1 2 3 4 Next >>