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1.
T Taki M Kawamoto H Seto N Noro T Masuda R Kannagi M Matsumoto 《Journal of biochemistry》1983,94(3):633-644
This study was to find out whether induction of special glycolipids or glycosyltransferases for glycolipid synthesis which might be involved in the cell functions occurred during the differentiation. Mouse myeloid leukemia cell line (M1-), the differentiated cells (M1+), and a subcloned cell line (Mm1) were used for this purpose. Gangliotriaosylceramide (GA2) was the major glycolipid component in M1- cells. As a result of differentiation of M1- into M1+ cells, globotriaosylceramide (CTH) was newly induced as the main glycolipid, while GA2 decreased to a minor component. GA2 was found to be the main glycolipid in Mm1 cells but no CTH was recognized. All precursor glycolipids and glycosyltransferases required to complete the biosynthetic pathway glucosylceramide (CMH) leads to lactosylceramide (CDH) leads to GA2 leads to gangliotetraosylceramide (GA1) leads to sialosylgangliotetraosylceramide (GM1b) were found in M1- and also in Mm1 cells. A galactosyltransferase activity for CTH synthesis from CDH increased 10 fold during the differentiation. The induction of CTH in M1+ cells could be attributed to the increase of the galactosyltransferase activity. Both CTH as a surface marker and the galactosyltransferase as an enzyme marker are proposed as valuable markers of differentiation in M1- cells. Besides the galactosyltransferase, N-acetylglucosaminyltransferease involved in the formation of lactotriaosylceramide (amino-CTH) increased up to 3 fold during the differentiation. The increase of the enzyme activity seemed to be responsible for the biosynthesis of lactoneotetraosylceramide (paragloboside) which appeared in M1+ cells. 相似文献
2.
Glycolipid and glycoprotein transport through the Golgi complex are similar biochemically and kinetically. Reconstitution of glycolipid transport in a cell free system 总被引:3,自引:2,他引:1
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《The Journal of cell biology》1990,111(2):421-428
Glycolipid transport between compartments of the Golgi apparatus has been reconstituted in a cell free system. Transport of lactosylceramide (galactose beta 1-4-glucose-ceramide) was followed from a donor to an acceptor Golgi population. The major glycolipid in CHO cells is GM3 (sialic acid alpha 2-3 galactose beta 1-4-glucose-ceramide). Donor membranes were derived from a Chinese hamster ovary (CHO) cell mutant (Lec2) deficient in the Golgi CMP-sialic acid transporter, and therefore contained lactosylceramide as the predominant glycolipid. Acceptor Golgi apparatus was prepared from another mutant, Lec8, which is defective in UDP-Gal transport. Thus, glucosylceramide is the major glycolipid in Lec8 cells. Transport was measured by the incorporation of labeled sialic acid into lactosylceramide (present originally in the donor) by transport to acceptor membranes, forming GM3. This incorporation was dependent on ATP, cytosolic components, intact membranes, and elevated temperature. Donor membranes were prepared from Lec2 cells infected with vesicular stomatitus virus (VSV). These membranes therefore contain the VSV membrane glycoprotein, G protein. Donor membranes derived from VSV-infected cells could then be used to monitor both glycolipid and glycoprotein transport. Transport of these two types of molecules between Golgi compartments was compared biochemically and kinetically. Glycolipid transport required the N- ethylmaleimide sensitive factor previously shown to act in glycoprotein transport (Glick, B. S., and J. E. Rothman. 1987. Nature [Lond.]. 326:309-312; Rothman, J. E. 1987. J. Biol. Chem. 262:12502-12510). GTP gamma S inhibited glycolipid and glycoprotein transport similarly. The kinetics of transport of glycolipid and glycoprotein were also compared. The kinetics of transport to the end of the pathway were similar, as were the kinetics of movement into a defined transport intermediate. It is concluded that glycolipid and glycoprotein transport through the Golgi occur by similar if not identical mechanisms. 相似文献
3.
K E Creek V P Walter D Evers E Yeo W L Elliott P F Heinstein D M Morré D J Morré 《Biochimica et biophysica acta》1984,793(2):133-140
Previous studies indicated a reproducible pattern of altered glycosphingolipid biosynthesis accompanying late stages of liver tumorigenesis in the rat induced by the carcinogen 2-acetylaminofluorene. The sequence began with a dramatic elevation in CMP-sialic acid:lactosylceramide sialyltransferase and was followed by sequential elevations and eventual depressions in other enzymes catalyzing sugar transfers to glycolipid acceptors. The present study focused on the early events of glycolipid biosynthesis during the first 11 weeks of 2-acetylaminofluorene administration according to the same feeding schedule as used previously. Transient elevations in CMP-sialic acid synthetase and elevations in neutral glycosphingolipid precursors to gangliosides were found to precede the major elevations in CMP-sialic acid:lactosylceramide sialyltransferase (GM3 synthetase) noted earlier. Two cycles of response were observed prior to the initiation of the sustained enhancement of biosynthesis of precursor ganglioside, GM3, and/or a significant increase in total or lipid-soluble sialic acid. In vitro rates of sialyl transfer from CMP-sialic acid to endogenous protein acceptors were not altered. The results suggest that the previous observations of altered ganglioside biosynthesis following 2-acetylaminofluorene administration are not an isolated occurrence but may represent late events in a sequence or 'cascade' of biochemical change involving, as well, biosynthesis of ganglioside precursors, CMP-sialic acid and neutral glycosphingolipids. 相似文献
4.
Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine 总被引:3,自引:0,他引:3
A Morita S Miura R H Erickson M H Sleisenger Y S Kim 《Biochimica et biophysica acta》1986,883(3):506-516
Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains. 相似文献
5.
Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance. 相似文献
6.
Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I 总被引:24,自引:0,他引:24
J D Esko J L Weinke W H Taylor G Ekborg L Rodén G Anantharamaiah A Gawish 《The Journal of biological chemistry》1987,262(25):12189-12195
We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells. 相似文献
7.
Norambuena L Marchant L Berninsone P Hirschberg CB Silva H Orellana A 《The Journal of biological chemistry》2002,277(36):32923-32929
The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-side-out Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi. In vitro assays using a Golgi-enriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1- MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed in S. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose. 相似文献
8.
Preparation and characterization of sonicated vesicles of various lipid composition containing hydroxy and normal fatty acid ceramides are reported. Such vesicles have been successfully used for the first time as acceptors for the assays of lipid glycosyltransferases, UDP-galactose:ceramide galactosyltransferase and UDPglucose: ceramide glucosyltransferase. Stability of the vesicles and the optimal enzyme activities were the criteria used to select the final composition of the vesicles. The activities of the glycosyltransferases were dependent not only on the appropriate assay conditions but also on the type and source of the phospholipids used to form the liposomes. Ceramides containing normal fatty acids were incorporated into phosphatidylcholine vesicles in a molar ratio of 1 : 3.4 and used as the acceptor for the assay of UDPglucose:ceramide glucostyltransferase. For the assay UDP-galactose:ceramide galactosyltransferase, vesicles were prepared by sonication of bovine brain ethanolamine phospholipids, phosphatidylcholine and ceramide containing alpha-hydroxy fatty acids, in a molar ratio of 6 : 0.57 : 1. The size of the vesicles as determined by electron microscopic measurement ranged mostly between 200--500 A. The results obtained by selective labelling of the outer surface amino groups with the membrane-impermeable reagent, 2,4,6-trinitrobenzenesulfonic acid, indicated that the ethanolamine phospholipid-containing liposomes consisted of closed vesicles. After incubation with the appropriate cofactors and labelled sugar nucleotides, the radioactive reaction products were shown to cochromatograph with the authentic standards by thin-layer chromatography and autoradiography. 相似文献
9.
Topography of glycosyltransferases involved in the initial glycosylations of gangliosides. 总被引:8,自引:0,他引:8
We attempted to establish within which organelle UDP-Glc:ceramide beta 1----1'glucosyltransferase (GlcT) is located and moreover to obtain information about its orientation on intracellular membranes as well as that of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) and CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase (SAT-1). An extremely purified Golgi apparatus fraction was the only liver fraction where a ceramide-dependent formation of glucosylceramide could be demonstrated. This Golgi fraction, mainly constituted by stacks of intact cisternae which retained the same topographical orientation as in vivo, was then incubated with liposomal dispersions of glycosphingolipid-glycosyltransferase acceptors in reaction mixtures containing all the requirements for enzyme activity but no detergent. Under such conditions, SAT-1 and other late acting glycosyltransferases were over 90% latent, while both GlcT and GalT-2 were just as active as in the detergent-containing assay; they were still inhibited by EDTA. Sepharose-immobilized ceramide and Sepharose-immobilized glucosylceramide were found to be suitable acceptors for GlcT and GalT-2, respectively, still using intact Golgi cisternae as the enzyme source. Moreover, a part of GlcT and GalT-2 activity was released from intact Golgi cisternae upon cathepsin D treatment. These results provide strong evidence that GlcT and GalT-2 face the cytoplasmic side of the Golgi apparatus, whereas SAT-1 and the other late acting enzymes face the luminal side. 相似文献
10.
We have previously engineered transgenic insect cell lines to express mammalian glycosyltransferases and showed that these cells can sialylate N-glycoproteins, despite the fact that they have little intracellular sialic acid and no detectable CMP-sialic acid. In the accompanying study, we presented evidence that these cell lines can salvage sialic acids for de novo glycoprotein sialylation from extracellular sialoglycoproteins, such as fetuin, found in fetal bovine serum. This finding led us to create a new transgenic insect cell line designed to synthesize its own sialic acid and CMP-sialic acid. SfSWT-1 cells, which encode five mammalian glycosyltransferases, were transformed with two additional mammalian genes that encode sialic acid synthase and CMP-sialic acid synthetase. The resulting cell line expressed all seven mammalian genes, produced CMP-sialic acid, and sialylated a recombinant glycoprotein when cultured in a serum-free growth medium supplemented with N-acetylmannosamine. Thus the addition of mammalian genes encoding two enzymes involved in CMP-sialic acid biosynthesis yielded a new transgenic insect cell line, SfSWT-3, that can sialylate recombinant glycoproteins in the absence of fetal bovine serum. This new cell line will be widely useful as an improved host for baculovirus-mediated recombinant glycoprotein production. 相似文献
11.
The major sialic acid containing glycolipid has been isolated from rat intestinal mucosa. Characterization of this ganglioside by thin layer and gas chromatographic analysis indicates that it is an hematoside (GM3) with the major portion of the sialic acid in the N-glycolyl form. The distribution of this ganglioside was determined in villus and crypt cells isolated from rat intestine. The hematoside content of crypt cells was found to be significantly decreased when compared to villus cells. CMP-sialic acid:lactosylceramide sialyltransferase, responsible for the sialylation of lactosylceramide, was measured in differentiated villus and undifferentiated crypt cells and found to be greatly reduced in the crypt cell fraction. The present study demonstrates that marked differences in ganglioside content and biosynthesis occur in contiguous populations of cells in varying states of differentiation when isolated from normal rat intestine. 相似文献
12.
—This report deals with some properties of the enzyme from rat brain, which catalyses in vitro the formation of lactosylceramide, a probable precursor of the gangliosides, from UDP-galactose and glucosylceramide. The enzyme is present in microsomes, mitochondria and synaptosomes, the latter having the highest specific activity, and appears to be firmly bound to the membranes of these particles. The enzymic activity is optimal at pH 6·8 and requires the presence of Mn2+. Lactosylceramide, the product of the reaction, was tentatively identified by its elution pattern from a Florisil column, its resistance to mild alkaline hydrolysis, thin-layer co-chromatography with authentic standards in six solvent systems, and location of the radioactivity in the galactose portion of the product obtained after incubation of labelled UDP-galactose with unlabelled glucosylceramide and in the glucose portion after incubation of glucose-labelled glucosylceramide with unlabelled UDP-galactose. The activity of this enzyme, per unit of brain weight, was found to be highest at birth and to decrease gradually thereafter. A similar age distribution was observed for another galactosyltransferase, the one which catalyses the formation of ganglioside GM1 from Tay-Sachs’ganglioside. In contrast, the activity of a third galactosyltransferase, which catalyses the formation of psychosine, and thus possibly is related to the increase of cerebrosides during myelination, is negligible during the first week and maximal about 20 days after birth. 相似文献
13.
Prior studies have demonstrated that sex hormones can influence the glycosphingolipid composition of different organs, including small intestine. However, to date, the effects of testosterone on glycosphingolipids of rat small intestinal mucosa have not been examined. Experiments were conducted to examine the effect of subcutaneous administration of synthetic testosterone (500 micrograms/100 g body wt.) on the gangliosides and neutral glycosphingolipids of rat small intestinal mucosa. Their results demonstrated that testosterone administrations: (i) increased the ganglioside content including hematoside (GM3); (ii) increased the total content of neutral glycosphingolipids, which was due to the increases in glucosylceramide and globotriaosylceramide; (iii) increased the activities of cytidine 5'-monophosphate-N-acetylneuraminic acid: lactosylceramide sialyltransferase, and UDPgalactose: lactosylceramide galactosyltransferase; (iv) increased the percentage of the long chain base phytosphingosine in hematoside, glucosyl-, and globotriaosylceramide; and (v) significantly altered the fatty acid composition of each of these glycosphingolipids. These results demonstrate that administration of testosterone induces alterations in glycosphingolipid composition and glycosyltransferases activities in rat small intestinal mucosa. 相似文献
14.
Localization in the Golgi apparatus of rat liver UDP-Gal:glucosylceramide beta 1----4galactosyltransferase 总被引:2,自引:0,他引:2
The presence and subcellular localization of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) was investigated in rat liver. For this purpose, purified Golgi apparatus, endoplasmic reticulum, and plasma membrane fractions were prepared from the liver and used as the enzyme source for detecting GalT-2. A pure Golgi apparatus, highly enriched in many glycosyltransferases, was the only fraction where GalT-2 was measurable. The reaction product formation rate under appropriate assay conditions, which requires high detergent concentration and Mn2+, was low but comparable with that of other glycosyltransferases. The product formation was stimulated by exogenously added acceptor GlcCer, donor UDP-Gal, and Golgi protein. The reaction product was a single spot that was identified by chromatographic behavior, sensitivity to beta-galactosidase, and permethylation studies as Gal beta 1----4Glc beta 1----1'Cer (lactosylceramide). A metabolic experiment, performed by determining the glycosphingolipids which became radioactive in the above subcellular fractions prepared from the liver of animals treated with glucose-labeled glucosylceramide, further indicated that the in vivo glycosylation of glucosylceramide takes place in the Golgi apparatus. 相似文献
15.
Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum
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Sprong H Degroote S Nilsson T Kawakita M Ishida N van der Sluijs P van Meer G 《Molecular biology of the cell》2003,14(8):3482-3493
UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex. 相似文献
16.
Enzymatic basis for a lectin-resistant phenotype: increase in a fucosyltransferase in mouse melanoma cells 总被引:2,自引:0,他引:2
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In the search for the biochemical basis of the control of glycosylation of cell surface carbohydrates, revertant clones were isolated from previously characterized wheat germ agglutinin-resistant clones of B16 mouse melanoma cells by selection for resistance to Lotus tetragonolobus lectin or to ricin. Comparison of the wheat germ agglutinin-resistant clones with the parent and revertant clones indicated that this phenotype was correlated with an increased sensitivity to the Lotus lectin, a 60- to 70-fold increase in alpha 1 leads to 3 fucosyltransferase activity and a decreased sialic acid content of the N-glycosidic chains of glycoproteins. The results suggest a novel type of control mechanism for lectin resistance, an increase in a glycosyltransferase activity. The presence of alpha 1 leads to 3 bound fucose on N-acetylglucosamine residues would interfere with the addition of sialic acid by alpha 2 leads to 3 linkages to galactose residues in the carbohydrate units, and this change could explain the resistance to wheat germ agglutinin and the increased sensitivity to the Lotus lectin. A change in a regulatory gene for the fucosyltransferase as a possible primary cause for the changed phenotype is discussed. 相似文献
17.
Lysolecithin markedly stimulated membrane-bound UDP-galactose:glycoprotein galactosyltransferase. The parent molecule lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidic acid, lysophosphatidic acid or glycerophosphorylcholine did not activate the enzyme suggesting that both fatty acyl- and phosphorylcholine groups are required for the enzyme activation. The dose-effect of lysolecithin showed sigmoidal kinetics and the Vmax of the enzyme was increased several-fold by lysolecithin. Saturating amounts of Triton masked the effect of lysolecithin. Pre-incubation with phospholipase A also activated the enzyme. A possible role of membrane lysolecithin is indicated in regulating the enzymes of glycoprotein synthesis. 相似文献
18.
Bina Toor Lilian McGregor John L. McGregor Serge Renaud Kenneth J. Clemetson 《生物化学与生物物理学报:生物膜》1984,770(2):178-182
Female rats were administered oral contraceptives and the levels of sialic acid on platelet membrane and granule glycoproteins were compared to controls using a sialic acid assay and a fluorescein-conjugated wheat germ agglutinin binding assay and also by measuring the binding of 125I-labelled wheat germ agglutinin to glycoprotein bands from platelets separated by polyacrylamide electrophoresis. The contraceptive-treated rats showed increased levels of glycoprotein sialylation which may partly explain the altered physiological function of the platelets. 相似文献
19.
K Furukawa J E Minor J D Hegarty V P Bhavanandan 《The Journal of biological chemistry》1986,261(17):7755-7761
The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes. 相似文献
20.
Ricardo O. Duffard 《Molecular and cellular biochemistry》1979,26(2):119-122
Summary Brain from mature rats has been shown previously to contain a natural inhibitor of rat brain sialyltransferase I (CMP-sialic acid: lactosylceramide sialyltransferase activity). This same inhibitor preparation was effective against sialyltransferase I and a second sialyltransferase activity from different lines of cultured cells. Cardiolipin which stimulates sialyltransferase I activity in cultured cells apparently was not required for inhibition. Inhibition was specific for sialyltransferase activities while a third ganglioside biosynthetic enzyme, UDP-gal: glucosyl-ceramide galactosyltransferase activity, was not inhibited. Inhibition of sialyltransferase I was linear with time, heat-resistant, and increased with inhibitor concentration.Gangliosides are sialic-acid containing glycosphingolipids found in brain as well as extraneural tissues and cultured cells t,2. Although the functions of gangliosides are unknown, they appear to play a role in morphological differentiation3 sensory and visual stimulation 4 and as receptor for cholera toxin5–8 and possibly thyroid stimulating hormone9. The monosaccharide units are added to the elongating oligosaccharide chain of the gangliosides in step-wise fashion and different glycosyltransferases catalyze each addition10,11. The activities of these enzymes have been observed to change during development12,14 malignant transformation13 and morphological differentiation3.Although little is known about the regulation of ganglioside synthesis, a natural inhibitor of CMP-AcNeu: GL-2 sialyltransferase (sialyltransferase I) from rat brain has been described14,15. As the inhibitor activity increased with the age of the animal, the same authors suggested that it may regulate the biosynthetic pathway of gangliosides. In this paper, the effects of the rat brain inhibitor on the activities of ganglioside biosynthetic enzymes from several cultured cell lines are described.Abbreviations AcNeu
N-acetylneuraminic acid
- GL1
glucosylceramide
- GL-2
lactosylceramide
- GM3
Nacetylneuraminylgalactosylglucosylceramide
- GM1
gal actosyl-N-acetylgalactosaminyl-(N-acetylneuraminylgalactosylglucosylceramide
- GD1a
Nacetylneuraminylgalactosyl-N-acetylgalactosaminyl-(Nacetylneuraminyl)-galactosylglucosylceramide.
DR. DUFFARD was a recipient of a Fogarty Center Fellowship. 相似文献