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1.
Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver 总被引:2,自引:0,他引:2
A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR. 相似文献
2.
Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9. 相似文献
3.
The demonstration of a precursor-product relationship in the course of GM1 and GD1a biosynthesis is described in the present paper. We injected rats with GM2 gangliosides [GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1'Cer] of brain origin, which were isotopically radiolabeled on the GalNAc ([GalNAc-3H]GM2) or sphingosine ([Sph-3H]GM2) residue. We then compared the time-courses of GM1 and GD1a biosynthesis in the liver after the administration of each radiolabeled GM2 derivative. After the administration of [GalNAc-3H]GM2, GM1, and GD1a were both present as doublets, that could be easily resolved on TLC. The lower spot of each doublet was identified as a species having the typical rat brain ceramide moiety and represented gangliosides formed through direct glycosylation of the injected GM2. The upper spot of each doublet was identified as a species having the typical rat liver ceramide moiety and represented gangliosides formed through recycling of the [3H]GalNAc residue, released during ganglioside catabolism. After the administration of [Sph-3H]GM2, only ganglioside with the rat brain ceramide moiety were found, that represented the sum of ganglioside formed through direct glycosylation and those formed through recycling of some sphingosine-containing fragments. In each case, the time-course of GM1 and GD1a biosynthesis exhibited a precursor-product relationship. The curve obtained from the direct glycosylation showed a timing delay with respect to those obtained from recycling of GM2 fragments. These results are consistent with the hypothesis that the sequential addition of activated sugars to a sphingolipid precursor is a dissociative process, catalyzed by physically independent enzymatic activities. 相似文献
4.
GM1 ganglioside beta-galactosidase (GM1-beta-galactosidase) was purified from normal cat brain and liver by a combination of classical and affinity procedures. The final preparation of brain GM1-beta-galactosidase was enriched over 2000-fold with a 36% yield. However, the product was shown to contain several components by disc gel electrophoresis. GM1-beta-galactosidase was also purified from liver with greater than a 30 000-fold enrichment and 40% yield. The liver enzyme was judged homogeneous by disc gel electrophoresis at pH 4.3, 8.1, and 8.9 and by gel chromatography. Both liver and brain GM1-beta-galactosidase(s) eluted as sharp symmetrical peaks from Sephadex G-200 with molecular weights of 250 000 +/- 50 000. The apparent Km determined for 4-methylumbelliferyl beta-D-galactopyranoside (4-MU-Gal) using partially purified brain GM1-beta-galactosidase was 1.73 X 10(-4) M. Liver GM1-beta-galactosidase gave a Km with 4-MU-Gal of 3.25 X 10(-4) M and for [3H]GM1 ganglioside a Km of 4.51 X 10(-4) M was calculated. The pH optima of brain and liver GM1-beta-galactosidase using 4-MU-Gal was 3.8-4.5. By contrast, liver GM1-beta-galactosidase gave a sharp activity peak at pH 4.2 with [3H]GM1 ganglioside. Inhibition by mercuric chloride and sensitivity to hydrogen peroxide and persulfate suggest the involvement of a sulfhydryl in catalysis. 相似文献
5.
Substrate specificity of GM2 and GD3 synthase of Golgi vesicles derived from rat liver 总被引:1,自引:0,他引:1
Several GM3 derivatives have been synthesized. Among them were lyso-GM3 derivatives and GM3 analogues with modifications in the sialic acid moiety. They were used as glycolipid acceptors in assays for GM2 and GD3 synthase of rat liver Golgi. Analysis of the resulting enzyme activities and of the reaction products revealed different substrate specificities for GM2 and GD3 synthase although the normal glycolipid acceptor for both transferases is ganglioside GM3. Specificity of GD3 synthase is strongly determined by the substrate's negative charge and the acyl residue in amide bond to the amino group of neuraminic acid, while GM2 synthase reacts quite indifferently to these changes in the sialic moiety of the substrate. Both enzymes seem to be sensitive to the spatial extension at the neuraminic acid's carboxylic group. 相似文献
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Oligosialogangliosides inhibit GM2- and GD3-synthesis in isolated Golgi vesicles from rat liver 总被引:2,自引:0,他引:2
H K Yusuf G Schwarzmann G Pohlentz K Sandhoff 《Biological chemistry Hoppe-Seyler》1987,368(5):455-462
The effect of end-product gangliosides (GD1a, GT1b, GQ1b) on the activities of two key enzymes in ganglioside biosynthesis, namely GM2-synthase and GD3-synthase in rat liver Golgi apparatus, has been investigated in detergent-free as well as in detergent-containing assays. In detergent-free intact Golgi vesicles, phosphatidylglycerol was used as a stimulant. This phospholipid was earlier shown to stimulate the activity of GM2-synthase without disrupting the vesicular intactness; it has, however, no effect on GD3-synthase (Yusuf, H.K.M., Pohlentz, G., Schwarzmann, G. & Sandhoff, K. (1983) Eur. J. Biochem. 134, 47-54). In the presence of this stimulant, all higher gangliosides inhibited the activity of GM2-synthase, the inhibition being more profound with increasing negative charge of the inhibiting gangliosides. These inhibitions are unspecific, but they do not exclude an end-product regulation of ganglioside biosynthesis. In detergent-solubilized Golgi membranes, on the other hand, the inhibition pattern was completely different. Here, ganglioside GD1a was the strongest inhibitor of GM2-synthase, followed by GM1 and GM2, but GT1b also inhibited this enzyme appreciably, in fact more strongly than GM1 or GM2. On the other hand, GQ1b had no effect at all. Conversely, GD3-synthase activity was most strongly inhibited by GQ1b, followed by GT1b, but GD1a also inhibited this enzyme almost as strongly as GT1b. These latter findings indicate that feed-back control of the a- and the b-series pathways of ganglioside biosynthesis is probably not specific, but the pathways appear to be inhibited more preferably by their respective end-products than by any other gangliosides of the same of the other series. 相似文献
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Cholesterol ester hydrolase activity was determined at 3 h time intervals over 24 h in lysosomes, cytosol and microsomes from ad libitum-fed and 24 h food-deprived female rat liver. Diurnal rhythms were identified for the acid and neutral esterases, which were strikingly changed by fasting. In fed animals, lysosomal esterase specific activity exhibited a peak at noon and a sustained medium rate at early darkness, whereas total esterase was maximal at midnight. The circadian patterns of the cytosolic and the microsomal esterases paralleled each other, though the amplitude of rhythms differed, showing higher activities around midnight. After fasting, cholesterol esterase activity from all cell fractions reached a maximum near dark onset. These results are the first to indicate that cholesteryl ester hydrolysis may play a role in generating the diurnal rhythm of hepatic cholesterol. 相似文献
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Two ribonuclease H activities have been purified from Saccharomyces cerevisiae. The major protein, RNase HA is an acidic protein with a molecular weight of 65,000. RNase HB is a basic protein with molecular weight of 54,000. Both RNases are active at alkaline pH range and require divalent cations for activity. RNase HA has an absolute requirement for Mg2+, while Mn2+ can replace Mg2+ for RNase HB. RNase HA is inhibited by low concentrations of N-ethylmaleimide, whereas RNase HB activity is unaffected under similar conditions. Substrate specificity studies using various polyribonucleotide · poly-deoxynucleotide hybrids showed that RNase HA preferentially degrades polycytidylate, while RNase HB is specific for polyadenylate. Kinetic analysis of the degradation of specifically end-labeled polymers and analysis of the products of the two yeast RNase H enzymes showed that yeast RNase HA is an endonuclease producing 5′-phosphorylated oligonucleotides while yeast RNase HB is a 5′-exonuclease producing 5′-AMP. 相似文献
13.
The enzymatic hydrolysis of the terminal galactose from GM1-ganglioside is monitored by a colorimetric procedure. The NADH generated from the oxidation of released galactose with NAD and galactose dehydrogenase is employed to reduce p-iodonitrotetrazolium and the absorbance of the product, p-iodonitrotetrazolium formazan, is measured. The method can detect as little as 0.5 nmol of galactose. Hydrolysis of GM1-ganglioside is accomplished using β-galactosidase from the marine gastropod Turbo cornutus. The enzymatic release of galactose is maximal at pH 3.5, and the reaction rate is linearly proportional to incubation time for 30 min, under the conditions employed. The presence of GM2-ganglioside in the reaction mixture, after hydrolysis has occurred, is demonstrated by thin-layer chromatography. 相似文献
14.
R Ghidoni M Trinchera B Venerando A Fiorilli S Sonnino G Tettamanti 《The Biochemical journal》1986,237(1):147-155
The pathways of metabolic processing of exogenously administered GM1 ganglioside in rat liver was investigated at the subcellular level. The GM1 used was 3H-labelled at the level of long-chain base ([Sph(sphingosine)-3H]GM1) or of terminal galactose ([Gal-3H]GM1). The following radioactive compounds, derived from exogenous GM1, were isolated and chemically characterized: gangliosides GM2, GM3, GD1a and GD1b (nomenclature of Svennerholm [(1964) J. Lipid Res. 5, 145-155] and IUPAC-IUB Recommendations [(1977) Lipids 12, 455-468]); lactosylceramide, glucosylceramide and ceramide; sphingomyelin. GM2, GM3, lactosylceramide, glucosylceramide and ceramide, relatively more abundant shortly after GM1 administration, were mainly present in the lysosomal fraction and reflected the occurrence of a degradation process. 3H2O was also produced in relevant amounts, indicating complete degradation of GM1, although no free long-chain bases could be detected. GD1a and GD1b, relatively more abundant later on after administration, were preponderant in the Golgi-apparatus fraction and originated from a biosynthetic process. More GD1a was produced starting from [Sph-3H]GM1 than from [Gal-3H]GM1, and radioactive GD1b was present only after [Sph-3H]GM1 injection. This indicates the use of two biosynthetic routes, one starting from a by-product of GM1 degradation, the other implicating direct sialylation of GM1. Both routes were used to produce GD1a, but only the first one for producing GD1b. Sphingomyelin was the major product of GM1 processing, especially at the longer times after injection, and arose from a by-product of GM1 degradation, most likely ceramide. 相似文献
15.
Jirkovská M Majer F Smídová J Stríteský J Shaik GM Dráber P Vítek L Marecek Z Smíd F 《Glycoconjugate journal》2007,24(4-5):231-241
(Glyco)sphingolipids (GSL) are believed to protect the cell against harmful environmental factors by increasing the rigidity
of plasma membrane. Marked decrease of membrane fluidity in cholestatic hepatocytes was described but the role of GSL therein
has not been investigated so far. In this study, localization in hepatocytes of a representative of GSL, the GM1 ganglioside,
was compared between of rats with cholestasis induced by 17α-ethinylestradiol (EE) and vehicle propanediol treated or untreated
animals. GM1 was monitored by histochemical reaction employing cholera toxin B-subunit. Our findings in normal rat liver tissue
showed that GM1 was localized in sinusoidal and canalicular hepatocyte membranes in both peripheral and intermediate zones
of the hepatic lobules, and was nearly absent in central zones. On the contrary, in EE-treated animals GM1 was also expressed
in central lobular zones. Moreover, detailed densitometry analysis at high magnification showed greater difference of GM1
expression between sinusoidal surface areas and areas of adjacent cytoplasm, caused as well by increased sinusoidal staining
in central lobular zone as by decreased staining in cytoplasm in peripheral zone. These differences correlated with serum
bile acids as documented by linear regression analyses. Both GM1 content and mRNA corresponding to GM1-synthase remained unchanged
in livers; the enhanced expression of GM1 at sinusoidal membrane thus seems to be due to re-distribution of cellular GM1 at
limited biosynthesis and could be responsible for protection of hepatocytes against harmful effects of bile acids accumulated
during cholestasis. 相似文献
16.
《FEBS letters》1994,350(2-3):219-222
The exposure of GM1 molecular species present in the native ganglioside, carrying C18:1 or C20:1 long-chain bases (LCB), to Dactylium dendroides galactose oxidase was studied. When native GM1 (49.3% C18:1 and 50.7% C20:1 LCB, respectively), was inserted in dipalmitoylphosphatidylcholine vesicles and partially oxidized (10%), the proportion of C18:1 and C20:1 species in the oxidized GM1 was 59.6% and 40.4%, respectively, suggesting a preferential action of the enzyme on the shorter species. The Vmax of the enzyme was higher on C18:1 GM1 than on C20:1 GM1. The molecular species were affected without any preference after partial (10%) oxidation of GM1 incorporated in egg phosphatidylcholine vesicles or in micellar form. These data indicate that the exposure of the terminal galactose moiety of GM1 ganglioside to galactose oxidase is affected by the ganglioside ceramide composition as well as the phospholipid environment, that presumably determine the distribution (molecular dispersion, segregation) of the ganglioside within the membrane. 相似文献
17.
Several intracellular proteins of low and intermediate molecular weights have been isolated from a variety of mammalian and plant tissues that possess an ability to catalyze the transfer or exchange of intact phospholipid molecules between different membrane systems. The soluble cytosolic fraction of the yeast Saccharomyces cerevisiae also contains phospholipid transfer activity that varies with both the state of cellular growth and the type of metabolic carbon source. This activity is protein in nature and very unstable, and requires powerful separation techniques for its purification. Here we report the isolation and characterization of two phospholipid transfer proteins from yeast, one of which we believe represents a partial proteolytic product of the other. The two proteins were purified to near homogeneity through a combination of dye-ligand and high performance ion-exchange chromatographic techniques. Transfer protein I (TP-I) is eluted at a lower ionic strength from an anion-exchange column than transfer protein II (TP-II), which reflects the difference in their isoelectric points; TP-I has a pI of 6.3, while that for TP-II is 6.1. Both species have the same apparent molecular weight of 33,400 and virtually identical substrate specificities. The order of the relative rates of phospholipid transfer are phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylserine. 相似文献
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《Biochimica et Biophysica Acta (BBA) - Enzymology》1978,522(1):258-266
Asparagine synthetase (L-aspartate: ammonia ligase (AMP-forming, EC 6.3.1.1) activity in rat liver increased when the animals were put on a low casein diet. The enzyme was purified about 280-fold from the supernatant of rat liver homogenate by a procedure comprising ammonium sulfate fractionation, DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration. The optimal pH of the enzyme was in the range 7.4–7.6 with glutamine as an amide donor. The molecular weight was estimated to be approximately 110 000 by gel filtration. Chloride ion was required for the enzyme activity. The apparent Km values for L-aspartate, L-glutamine, ammonium chloride, ATP, and Cl− were calculated to be 0.76, 4.3, 10, 0.14, and 1.7 mM, respectively. The activity was inhibited by l-asparagine, nucleoside triphosphates except ATP, and sulflhdryl reagents.It has been observed that the properties of asparagine synthetase from rat liver are not different from those of tumors such as Novikoff hepatoma and RADA 1. 相似文献
20.
Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside GM3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside GM3 as well as for ganglioside GD3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside GM3. Based on this idea we used a variety of GM3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for GD3 synthase (of rat liver Golgi vesicles). This study revealed that those GM3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (GD3 synthase). This may indicate similarities in the substrate binding sites of these enzymes. 相似文献