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1.
A fluorometric method for monitoring the enzymic hydrolysis of the terminal galactose from GM1-ganglioside has been developed. The released galactose is oxidized with galactose dehydrogenase and NAD and the fluorescence of the product NADH measured. This method can detect as little as 0.1 nmol of galactose. β-Galactosidase from the gastropod Turbo cornutus was employed for the hydrolysis reaction. The rate of GM1-ganglioside hydrolysis is linearly proportional to incubation time for 30 min under the assay conditions employed. In addition to galactose, the other product of hydrolysis, GM2-ganglioside, is identified by thin-layer chromatography. This procedure provides a convenient and specific method for measuring the release of galactose from GM1-ganglioside.  相似文献   

2.
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   

3.
When entrapped into liposomes composed of phosphatidylcholine and other lipids, β-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) purified from Aspergillus oryzae could cleave the β-galactosidic bond of the terminal galactose of galactocerebroside and GM1-ganglioside (II3NeuAc-GgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosylglucosylceramide), while the free enzyme could not. The products of the hydrolysis of galactocerebroside were found to be β-galactose and ceramide, which was confirmed by using a fluorescent analog of galactocerebroside, 1-O-galactosyl-2-N-(1-dimethylaminonaphthalene-5-sulfonyl)-sphingosine, as substrate. The formation of GM2-ganglioside (II3NeuAc-GgOse3Cer, N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosylglucosylceramide) by the hydrolysis of GM1-ganglioside was also demonstrated. The lipid composition of the liposomes influenced the amount of the enzyme entrapped and the activity of the trapped enzyme. A large amount of the enzyme was entrapped into the liposomes composed of phosphatidylcholine-cholesterol-stearoylamine (molar ratio, 7:2:1). The enzyme trapped in the liposomes and that in those of phosphatidylcholine-cholesterol-sulfatide (molar ratio, 7:2:1) had higher activity on galactocerebroside and GM1-ganglioside than that in other liposomes. The activity of β-galactosidase trapped in liposomes was increased in the presence of detergent, while that of the free enzyme was not changed.By a similar procedure to introduce enzymes into hydrophobic environments, enzymes other than β-galactosidase might come to possess different substrate specificities.  相似文献   

4.
Ganglioside GM1 beta-galactosidase: studies in human liver and brain   总被引:10,自引:0,他引:10  
A microcolumn assay for ganglioside GM1 β-galactosidase (EC 3.2.1.23) has been developed using GM1 tritiated exclusively in the terminal galactose residue. The reaction is stimulated up to 100-fold by anionic and cationic detergents; this stimulation is inhibited by neutral detergents. 4-Methylumbelliferyl β-d-galactopyranoside is hydrolyzed about seven times more rapidly than GM1 in human brain (gray matter) and liver. Agarose gel filtration separated two forms of GM1 β-galactosidase in both brain and liver. The major form (ganglioside GM1 β-galactosidase A) had a molecular weight of 60–70 × 103 and the minor form (ganglioside GM1 β-galactosidase B) 600–800 × 103. The liver and brain GM1 β-galactosidases and 4-methylumbelliferyl β-galactosidase A cochromatographed on fractionation. The two forms of the enzyme in liver isolated by gel filtration corresponded to the two major forms found on starch gel electrophoresis and were converted to electrophoretically slower-moving forms after treatment with neuraminidase (EC 3.2.1.8, Cl. perfringens) suggesting that both are sialylated glycoproteins. The activity of GM1 β-galactosidase in the brain and liver tissue of patients with GM1 gangliosidosis Types I and II was less than 2% of control values. The mutation in each GM1 gangliosidosis appears to result in a severe reduction of activity of two ganglioside GM1 β-galactosidases.  相似文献   

5.
Biochemical studies in cat and human gangliosidosis   总被引:2,自引:0,他引:2  
The biochemical analysis of the hereditary neurological disease found in a family of Siamese cat is reported. The accumulation of GM1ganglioside in the brain was noted. Several glycosidase activities of these cat brains were compared with that of human gangliosidoses (Tay-Sachs disease and GM1-gangliosidosis). Glycosidase activities were estimated using ρ-nitrophenyl-glycosides, glucosyl-, galactosyl-ceramide and GM1-ganglioside as substrates. The results indicated the defect of the β-galactosidase activities for the ρ-nitrophenyl-β-galactoside and GM1-ganglioside in both cat and human GM1-gangliosidoses. Glycosidase activities for glucosyl- and galactosyl-ceramide were not changed in either gangliosidoses.  相似文献   

6.
Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM1-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar Km and V values; the more acidic form (designated β-galactosidase N1) was more heat stable than the other form (designated β-galactosidase N2). Antibodies evoked against the N1 and N2 β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.  相似文献   

7.
Immunization of mice with a synthetic GM3-lactam-BSA (bovine serum albumin) conjugate (designed to emulate the corresponding natural GM3-lactone conjugate), followed by fusion of splenocytes with myeloma cells, gave rise to more than 300 monoclonal hybridomas producing antibodies to GM3-lactam-BSA, which did not react with Glc-BSA and BSA. Eight antibody clones were randomly chosen from the positive 300 hybridomas. The eight clones, all belonging to the IgG class, were unreactive against GM3-ganglioside, whereas two antibodies (P5-1 and P5-3, both IgG1, ) reacted with GM3-ganglioside lactone. Binding of these two antibodies to the GM3-lactam-BSA conjugate was inhibited by soluble glycosides of GM2-, GM3-, and GM4-lactam and by GM3- and GM4-lactam, respectively, but not by Gb3 or asialo-GM1 and GM2-saccharides. A third antibody (P3; IgG2b, ) was inhibited by GM2-, GM3-, and GM4-lactam, but did not recognize GM3-ganglioside lactone.  相似文献   

8.
Abstract: Ganglioside composition of rat trigeminal nerve was studied during development in order to understand the changes that occur as a result of cellular differentiation in the nerve. The ganglioside composition of the trigeminal nerve was entirely different from that of brain. The major gangliosides in adult trigeminal nerve were GM3, GD3, and LM1 (sialosyl-lactoneotetraosylceramide or sialosylparagloboside). The structure of LM1 and other gangliosides was established by enzymatic degradation and by analysis of the products of acid hydrolysis. At 2 days after birth, when the Schwann cells were immature, GM3 and GD3 were the major gangliosides in the nerve, 50 and 18 mol %, respectively. As the nerve developed and Schwann cells proliferated and myelinated the axons, the mol % of GM3 and GD3 reduced and that of LM1 steadily increased. Polysialogangliosides did not change drastically with nerve development. The rate of deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids, cerebrosides, and sulfatides; thus, deposition appears to be localized mainly in the rat nerve myelin. LM1 also had long-chain fatty acids 22:0 and 24:0, which are not usually found in CNS gangliosides. The ganglioside pattern of the rat trigeminal nerve was very similar to that of rat sciatic nerve, but was different from that of rabbit and chicken sciatic nerve. The activity of the two key enzymes involved in the metabolism of GM3, viz., CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase and UDP-N-acetylgalactosamine:GM3-N-acetylgalactosaminyltransferase, was also studied during development of the nerve and brain. The developmental profiles of both enzymes were consistent with the amounts of GM3 present in the nerve.  相似文献   

9.
d-Gluconamide, d-gluconyl hydrazide, and N-(6-aminohexyl)-d-gluconamide were prepared from d-glucono-1,5-lactone by treatment with ammonia, hydrazine, and 1,6-diaminohexane, respectively. These d-gluconamide derivatives were tested for their inhibitory action on human liver lysosomal glucocerebrosidase and human spleen neutral aryl β-glucosidase. Analogous d-galactonamide derivatives were evaluated for their inhibition of human spleen galactocerebrosidase and GM1-ganglioside β-galactosidase. d-Gluconyl hydrazide and d-gluconamide were effective inhibitors of the lysosomal glucocerebrosidase, attaining 50% inhibition at 5 and 12 mm, respectively. In contrast, N-(6-aminohexyl)-d-gluconamide did not inhibit the glucocerebrosidase. d-Gluconyl hydrazide was also the most effective inhibitor of human liver and spleen aryl β-glucosidase, 50% inhibition being achieved at 4 mm concentration (competitive inhibition, Ki = 0.4–0.9 mM). d-Galactonamide was the most effective inhibitor of spleen galactocerebrosidase; 4 mm d-galactonamide caused 50% inhibition of the enzyme activity (noncompetitive inhibition). N-(6-Aminohexyl)-d-galactonamide is a potent inhibitor (90% inhibition, 5 mm) of GM1-ganglioside β-galactosidase but is without effect on galactocerebrosidase. It has, therefore, the potential usefulness in distinguishing between two of the galactosphingolipid β-galactosidases.  相似文献   

10.
Thermoresistant, recombinant β-galactosidase from Thermotoga maritima was purified and immobilized on the surface of epoxy-coated magnetic beads. The enzyme, which has hexameric quaternary structure as shown by gel filtration chromatography, attaches to the resin through multiple covalent linkages that involve different subunits. The bound enzyme shows higher stability than the free form. The immobilized enzyme showed to be efficient for the hydrolysis of lactose and the biosynthesis of galactooligosaccharides (GOS). The chemical structure of synthesized GOS has been determined by NMR revealing that the main product was β-3′-galactosyl lactose. Although β-galactosidases from different sources have been used for the same purposes, the distinct advantage of the methodology described in this communication is that the enzyme can be easily produced, purified and immobilized in large quantities.  相似文献   

11.
This study characterized a glycoside hydrolase family 42 (GH42) β-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 β-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 β-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 β-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 β-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 β-galactosidases formed structurally similar GOS with varying preferences.  相似文献   

12.
Formation and turnover of myelin ganglioside   总被引:7,自引:6,他引:1  
—In young adult rats, the formation and turnover of GM1-ganglioside in myelin were compared with the formation and turnover of GM1-ganglioside in whole brain and of total lipids in whole brain and myelin, after injection of d-[1-14C]glucosamine. During the first 24 hr after injection, the specific activity of GM1-ganglioside in myelin was less than 25 per cent of that of GM1-ganglioside in whole brain. The specific activity of ganglioside in whole brain was maximal at 24 hr and then declined steadily during the next 3 months, whereas the specific activity of GM1-ganglioside in myelin continued to increase and did not reach a peak until about one month after injection, by which time its specific activity had increased five-fold. Consequently, the specific activity of GM1-ganglioside in myelin was 50 per cent higher than ganglioside in whole brain after one month. These differences in the formation and turnover of GM1-ganglioside in myelin and of whole brain are similar to those of other lipids of myelin and of whole brain, indicating that the metabolic activity of myelin ganglioside is similar to myelin lipids, but differs from whole brain lipids or whole brain gangliosides. These data provide additional evidence that ganglioside in myelin is an intrinsic constituent of the myelin sheath. GT1 (G1), GD1b, (G2), GD1a (G3), GM1 (G4), GM2 (G5), GM3 (G6).  相似文献   

13.
The regioselectivity of β-galactosidase derived from Bacillus circulans ATCC 31382 (β-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for β-1,3-galactosidase but also for β-galactosidases of different origins. The relative hydrolysis rate of Galβ-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of β-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of β-1,3-, β-1,4- and β-1,6-linked D-galactosyl-D-mannose was subsequently established.  相似文献   

14.
A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 UoNPG/mg protein. The Km, kcat and kcat/Km values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 38 mM, 20 s-1, 530 M-1·s-1 and 1.67 mM, 540 s-1, 325 000 M-1·s-1, respectively. The temperature optimum of β-galactosidase activity was 60–65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg2+ ions enhanced both activity and stability significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1→3) and β-(1→6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1→6)-D -Glc, β-D-Galp-(1→3)-D -Glc, β-D -Galp-(1→6)-D -Gal, β-D -Galp-(1→3)-D -Gal, and the trisaccharides β-D -Galp-(1→3)-D -Lac, β-D -Galp-(1→6)-D -Lac.  相似文献   

15.
The concurrent purification of the activator protein for sulphatide hydrolysis and for GM1-ganglioside hydrolysis including chromat ofocusing and hydrophobic chromatography stages is described. The purified preparation has a pl of 4.2 and the sub-unit Mr is 10 000. The stoichiometry of binding of sulphatide and ganglioside to the protein is very similar. Both activities are removed in similar proportions on binding to IgG purified from antisera raised against the activator protein. The probable identity of the activator protein for sulphatide hydrolysis with that for GM1-ganglioside hydrolysis and a molecular explanation for this identity are discussed.  相似文献   

16.
Non-acid and acid glycosphingolipids were isolated from feces of one litter of germ-free rats from day 17 to day 51. Quantitative and qualitative changes described for small intestine of conventional rats [Bouhours D, Bouhours J-F (1981) Biochem Biophys Res Commun 99:1384–89] were also found in the feces of these germ-free rats. A decrease in lactosylceramide and sialyllactosylceramide excretion and a change fromN-acetylneuraminic acid toN-glycoloylneuraminic acid, as well as an appearance of type 1 chain blood group H-active penta- and decaglycosylceramides were observed during the weaning period. Thus the dramatic changes seen in rat intestinal glycosphingolipids postnatally seem to be primarily regulated by non-microbial factors.Abbreviations GM3 GM3-ganglioside, II3NeuAc-LacCer or II3NeuGc-LacCer - SPG IV3NeuAc-nLcOse4Cer - GM1 GM1-ganglioside, II3NeuAc-GgOse4Cer  相似文献   

17.
The midgut of Dysdercus peruvianus is divided into three main sections (V1-V3) and is linked through V4 to the hindgut. The distribution of α-galactosidase activity in the different gut segments of D. peruvianus females was studied. α-galactosidase hydrolyzes the trisaccharide raffinose, the major carbohydrate of cotton seeds, on which the insects live. In D. peruvianus midgut, α-galactosidase activity is mainly found in soluble fractions of V1 contents. However, a comparison between specific activities using different α-galactosidase substrates in cotton seed extracts, V1 tissue homogenate, and midgut contents suggested that the contribution of the enzymes from seeds may be very significant. Gel filtration on Sephacryl S-200 of samples from seed extracts, V1 tissue, and V1 contents revealed that in all samples raffinose hydrolysis is accomplished by α-galactosidases with similar Mr (30,000 ± 3,000) and does not involve the activity of a β-fructosidase. Thermal inactivation studies of extracts from the three sources suggested that there was only one molecular form of the insect α-galactosidase and that the activity found in V1 contents includes enzymes derived from the seed kernel. In insects fed with cotton seeds, the α-galactosidase activity increased in parallel with diet ingestion. In starved insects fed with tablets of sucrose plus raffinose, an increase in α-galactosidase activity was also observed, confirming that the insect is able to synthesize part of the gut enzyme. The results indicated that raffinose digestion starts in V1 utilizing α-galactosidases derived from the seed kernel and by an additional α-galactosidase synthesized by insect tissues. The action of α-galactosidases liberates galactose and sucrose, which are sequentially hydrolyzed by the major membrane-bound α-galactosidase releasing glucose and fructose in V1 and V2 lumina. Arch. Insect Biochem. Physiol. 34:443–460, 1997. © 1997 Wiley-Liss, Inc.  相似文献   

18.
p-Hydroxybenzoyl β-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of β-galactosyl ester linkage by β-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl β-galactoside (pNP-Gal), a usual substrate with a β-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that β-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H2 18O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.  相似文献   

19.
Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM1, GM2, and GM3), di- (GD1a), and trisialogangliosides (GT1) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.  相似文献   

20.
Optical resolution of β-(1-naphthyl)alanine and β-(2-naphthyl)alanine have been efficiently carried out through enzymatic hydrolysis of their methyl ester and/or N-acetyl ester derivatives by immobilized enzymes. Difficulties related to the lipophilic character of these amino acids were overcome by using emulsions of n-butyl acetate–water as reaction medium. The use of an automatic recirculating apparatus allowed reproducible and repetitive use of the immobilized biocatalysts.  相似文献   

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