首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
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.
1. GM(1)-ganglioside, specifically tritiated in the terminal galactose, was hydrolysed by two forms of ;acid' methylumbelliferyl beta-galactosidase isolated on gel filtration. 2. Identification of GM(1)-ganglioside beta-galactosidase activity with the ;acid' methyl-umbelliferyl beta-galactosidases was based on the following: coincident elution profiles on gel filtration; simultaneous inactivation by heat and other treatments; stabilization of both activities by chloride ions; mutual inhibition of hydrolysis by the two substrates. 3. The two isoenzymes (I) and (II) showed general requirements for a mixture of anionic and nonionic detergents in the hydrolysis of the natural substrate. 4. Isoenzyme (I) differed from (II) in molecular size, pH-activity profile, relative resistance to dilution and in sensitivity to various inhibitors. 5. The most significant difference between the isoenzymes is in substrate saturation kinetics: (I) was hyperbolic whereas (II) was sigmoid. The apparent Michaelis constants were 28mum for (I) and 77mum for (II). Isoenzyme (I) was insensitive to GM(2)-ganglioside whereas (II) was inhibited, consistent with the hypothesis that GM(1)-ganglioside (and its analogue) acts as modifier in isoenzyme (II) but not in (I). 6. Isoenzyme (I) was membrane-bound whereas (II) was soluble; the former probably represents isoenzyme (II) bound to membrane components, thereby becoming activated. 7. Membranes may serve a dual role in enzyme catalysis involving lipids: as a medium where both enzyme and substrate are effectively concentrated, and as actual activator of enzymes through binding of the latter to specific membrane components.  相似文献   

4.
1. Human hepatic "acid" beta-galactosidase preparations, which had been purified approximately 250-fold, were examined for activities toward 4-methylumbelliferyl beta-galactoside, galactosylceramide, lactosylceramide, galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosyl-glucosylceramide (GM1-Ganglioside) and galactosyl-Cacetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM1-ganglioside). 2. The enzyme was active toward the synthetic substrate, GM1-ganglioside and asialo GM1-ganglioside but was inactive toward galactosylceramide. Under our assay conditions, optimized for lactosylceramidase II, the preparations were as active toward lactosylceramide as toward GM1-ganglioside or its asialo derivative. Teh apparent Km values for the three natural substrates were similar. When determined by the assay system of Wenger, D.A., Sattler, M., Clark, C. and McKelvey, H. (1974) Clin. Chim. Acta 56, 199-206, lactosylceramidecleaving activity was 0.2% of that determined by our assay system. This confirmed our previous suggestion that the Wenger assay system determines exclusively the activity of lactosylceramidase I, which is probably identical with galactosylceramide beta-galactosidase. 3. Crude sodium taurocholate was far more effective than pure taurocholate in stimualting hydrolysis of the three glycosphingolipids by the beta-galactosidase. However, crude tauroxycholate, suggesting that the unique activating capacity of the crude taurocholate might be due to taurodeoxycholate present as the major impurity. 4. Cl- was generally stimulatory for hydrolysis of the natural glycosphingolipids by our enzyme preparation. Effects of additional oleic acid and Triton X-100 Were generally minor in either direction. 5. When the enzyme preparation was diluted with water, activity toward the synthetic substrate declined rapidly while those toward the natural substrates were essentially stable. Activity toward the synthetic substrate remained much more stable when the enzyme was diluted with 0.1 M sodium citrate/phosphate buffer, pH 5.0. 6. These observations provide insight into the complex relationship among the human hepatic beta-galactosidases.  相似文献   

5.
Studies were done on the effect of bile salts on the rates of hydrolysis of the N-acetylneuraminyl linkages of several sialic acid-containing compounds by the neuraminidase of Clostridium perfringens. When GM3-ganglioside, two glycolipids (glycophorin and orosomucoid) and neuraminyl-lactose were used as substrates, hydrolysis was obtained even in the absence of bile salts, but addition of this detergent, below its critical micellar concentration, increased the reaction rates; above the critical micellar concentration of the detergent rates decreased again. When a second ganglioside, GM1, was used as substrate, the requirement for bile salts was absolute; hydrolysis was not observed at all without this detergent. With increasing concentrations of bile salt and in the presence of high concentrations of enzyme, rates of hydrolysis increased, reaching maximal values at fixed ratios of bile salt to GM1-ganglioside. Physical measurements showed that mixtures of bile salt and GM1-ganglioside form mixed micelles that have a higher critical micellar concentration, a lower molecular weight and greater axial ratio than the corresponding micelles of pure GM1-ganglioside.  相似文献   

6.
Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea.  相似文献   

7.
Although Gaucher's disease occurs in three distinct forms with greatly varying degrees of severity, there is no correlation between the clinical course of the disease and levels of residual glucocerebrosidase, the fundamental enzymatic deficiency. In an effort to study secondary changes which might contribute to the pathology of Gaucher's disease, homogenates of spleen, liver, and brain tissue, as well as serum from patients with Gaucher's disease were analyzed for their content of a number of lysosomal enzymes. Extracts of 8 Gaucher spleens contained 3- to 4-fold increases in acid phosphatase activity as well as 5-to 10-fold increases in galactocerebrosidase5 activity. The marked elevation in galactocerebrosidase activity in Gaucher spleen was documented using various [3H]galactose labeled galactocerebrosides as substrates and with [3H]galactose labeled lactocerebroside under the “lactosylceramidase I”5 assay conditions established by Suzuki (Tanaka, H., and Suzuki, K., 1975, J. Biol. Chem., 250, 2324–2332) that measure galactocerebrosidase activity specifically in the presence of Gmi-ganglioside β-galactosidase. Acid phosphatase determinations using extracts of liver from a case of infantile, neuropathic Gaucher's disease revealed a 2-fold elevation in this activity, whereas brain acid phosphatase activity in this case was similar to that of control tissue. Separation of hexosaminidase A and B activities on DEAE-Sephadex columns indicated increases in both forms of the enzyme in Gaucher tissue with the major increase occurring in the hexosaminidase B component. Glucuronidase and nonspecific esterase were observed to be elevated approximately 2-fold. However, not all lysosomal enzyme activities were increased. Levels of splenic arylsulfatase A and B, α-arabinosidase, sphingomyelinase, α-mannosidase, and Gmi-ganglioside β-galactosidase activities in Gaucher spleen were unremarkable. Gmi-ganglioside β-galactosidase was measured using 4-methylumbelliferyl-β-d-galactopyranoside and [3H]galactose labeled lactocerebroside under the specific assay conditions described by Suzuki for the determination of “lactosylceramidase II” activity. Although levels of arylsulfatase A and B in Gaucher spleen were similar to those of control tissue, arylsulfatase A activity was markedly reduced (20% of control) in homogenates of brain from the case of infantile (type 2) Gaucher's disease. The metabolic and pathologic consequences of these changes in lysosomal enzymes in Gaucher's disease are discussed.  相似文献   

8.
beta-Galactosidase from T. cornutus was resolved into two activity peaks by gel filtration column chromatography. The pH optima of the two peaks designated P1 and P2, were 5.5 and 3.0, respectively, when p-nitrophenyl-beta-D-galactopyranoside was used as the substrate. The molecular weights of P1 and P2 were 700,000 +/- 70,000 and 78,000 +/- 7800, respectively, when estimated by gel filtration chromatography. The activities of both forms of the enzymes are stimulated by anions such as Cl-, Br- and NO-3. While the activity of P1 was stimulated by low anion concentrations, P2 requires 700 times higher anion concentration for similar enhancement of activity. P1, the high molecular weight form hydrolyzes mainly galactose from small molecular weight beta-galactosides, such as p-nitrophenyl-beta-D-galactopyranoside, 4-methylumbelliferyl-beta-D-galactopyranoside, lactose, lactosylceramide and 3-O-beta-D-galactopyranosyl-D-arabinose, whereas P2, the low molecular weight form cleaves, in addition, all the beta-galactosides tested, including 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, GM1-ganglioside, asialo-GM1-ganglioside, asialo fetuin, alpha 1-acid glycoproteins and the tryptic peptides of the glycoproteins. The optimal conditions for the hydrolysis of the terminal galactose from GM1-ganglioside which does not occur in gastropods, such as T. cornutus, was found to require 40 mM NaCl and 1 mM sodium taurodeoxycholate at pH 3.0 in 50 mM sodium citrate buffer, conditions similar to those by mammalian beta-galactosidase.  相似文献   

9.
The carbohydrate composition was determined for ceramide hexosides isolated from brains of patients with Tay-Sachs disease and generalized gangliosidosis (hereby named GM1-gangliosidosis). Gray matter of patients with each disease showed a characteristic abnormal ceramide hexoside pattern. In Tay-Sachs gray matter, ceramide trihexoside is the major component, whereas ceramide tetrahexoside is barely detectable. In GM1-gangliosidosis, ceramide tetrahexoside is the major ceramide hexoside, while ceramide trihexoside is present only in small amount. These two major components have been characterized as the asialo derivatives of, respectively, the "Tay-Sachs ganglioside" (GM2-ganglioside) and the normal major monosialoganglioside (GM1-ganglioside). In both diseases, more than half the ceramide monohexoside of gray matter was glucocerebroside. Gray matter ceramide dihexoside, present in both diseases at higher than normal levels, was mostly ceramide lactoside, with possibly a small amount of ceramide digalactoside. Sulfatide contained only galactose. The abnormal ceramide hexoside pattern is limited to gray matter: white matter showed normal ceramide hexosides, i.e. a preponderance of monohexosides and sulfatide, with no detectable glucocerebroside.  相似文献   

10.
An assay method for glycosphingolipid glycosyltransferase activity using simple Sephadex G-50 gel permeation chromatography in an aqueous solvent has been developed. An acceptor glycosphingolipid and a donor radioactive nucleotide sugar were incubated with an enzyme source. The reaction mixture was loaded onto a Sephadex G-50 column previously equilibrated with 0.3% (w/v) Triton X-100, 0.1 M sodium chloride, and 0.02% (w/v) sodium azide. The radiolabeled reaction product was eluted by the same solvent in the excluded volume and was collected directly into a liquid scintillation vial, separated from other radioactive compounds. This assay method was utilized to determine the activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:GM3 ganglioside sialyltransferase, which catalyzes the synthesis of GD3 ganglioside, and proved to be as reliable and sensitive as previously published assay procedures. In addition, this assay can be carried out in less time and is simpler than previously reported procedures.  相似文献   

11.
To clarify the relationship between hexosaminidase A (HEX A) activity and GM2-ganglioside hydrolysis in atypical clinical situations of HEX A deficiency, we have developed a simple method to assess GM2-ganglioside metabolism in cultured fibroblasts utilizing GM2 labeled with tritium in the sphingosine portion of the molecule. The radioactive lipid is added to the media of cultured skin fibroblasts, and after 10 days the cells are thoroughly washed, then harvested, and their lipid composition analyzed by HPLC. The degree of hydrolysis of the ingested GM2 is determined by comparing the amount of radioactive counts recovered in undegraded substrate with total cellular radioactivity. A deficiency in GM2-ganglioside hydrolysis was demonstrated in seven HEX A-deficient adults with neurological signs and in two healthy-appearing adolescents with older affected siblings. In each case, an analysis of endogenous monosialoganglioside composition revealed an increase in GM2-ganglioside, confirming the presence of a block in the metabolism of GM2. No defect in GM2-catabolism was found in four other healthy individuals with HEX A deficiency. This method of assay is especially helpful in the evaluation of atypical cases of HEX A deficiency for the definitive diagnosis of GM2-gangliosidosis.  相似文献   

12.
A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.  相似文献   

13.
Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB(5) (the B subunit pentamer of cholera toxin) is investigated. When CtxB(5) was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t(12) approximately 8 min) and to migrate as SDS-stable pentamers (t(12) approximately 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of approximately 8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB(5) reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.  相似文献   

14.
Galactosyltransferase and 5'-nucleotidase were assayed in the same reaction mixture, with ovalbumin as exogenous acceptor of (14-C)galactose and with (3-H)AMP as the substrate for the 5'-nucleotidase assay. The substrates and reaction products of either assay had no significant effect on the activity of the other enzyme.  相似文献   

15.
An enzyme that transfers galactose from UDP-Gal to ganglioside GM2 (Tay-Sachs ganglioside) was concentrated 50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents or phospholipids as dispersing agents. Of the numerous detergents tested, sodium taurocholate and Triton CF-54 were most effective in stimulating the reaction. Cardiolipin alone was more effective than any of the detergents tested in stimulating enzyme activity. The pH optimum for the reaction varied with the nature of the dispersing agent. With sodium taurocholate, Triton CF-54 and cardiolipin, the pH optima were 6.2, 5.9, and 5.6, respectively. The enzyme had a nearly absolute requirement for Mn2+, with maximum activity being attained at a concentration of 15 mM Mn2+. Other divalent or trivalent cations were either less effective than Mn2+ or inhibited the transferase reaction. The Km values calculated for UDP-Gal and GM2 were 1.1 X 10(-4) M and 9.9 X 10(-5) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane and not part of the luminal contents. The newly synthesized GM2, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.  相似文献   

16.
An enzymatic determination method for galactosylceramide galactosidase (EC 3.2.1.46) was devised by using an enzymatic amplification reaction, NAD cycling. Galactose released by crude enzyme samples (tissue homogenates and cell suspensions) from galactosylceramide quantitatively reduced NAD to NADH by the galactose dehydrogenase reaction; then the NADH was amplified 6000-10,000-fold by NAD cycling and determined fluorometrically. A higher sensitivity of assay was obtained compared with the previous radiometric method. The present method was successfully applied to tissues from patients with Krabbe's disease, whose organs are deficient in galactosidase. The galactosidase reaction rate with a crude sample was not proportional to its concentration. However, the double-reciprocal plot of the reaction rate against the sample concentration became linear and provided a unique value of specific activity to each sample.  相似文献   

17.
The galactose oxidase-sodium borohydride method was used to specifically label the terminal N-acetylgalactosamine of three glycosphingolipids, Gm2-ganglioside, asialo-Gm2-ganglioside, and globoside. All of the compounds showed a minimum of 95% radiopurity, and generally more than 90% of the total radioactivity was located in the terminal galactosamine moiety. Globoside and asialo-Gm2-ganglioside were labeled to high specific activities comparable with those of the sphingolipids with a terminal galactose moiety, labeled with the same procedure. These labeled compounds were well suited as substrates for the study of specific sphingolipid N-acetylgalactosaminidase. Gm2-ganglioside, however, was a poor substrate for galactose oxidase, and its specific activity was only a small percentage of the others. Furthermore, because of the low specific activity of the galactosamine moiety, it was necessary to pretreat Gm2-ganglioside with unlabeled sodium borohydride to reduce the nonspecific labeling of other portions of the molecule. The use of labeled sodium borohydride of a very high specific activity may yield specifically labeled Gm2-ganglioside suitable for metabolic studies. Thus, the method is useful for labeling not only terminal galactose but also terminal N-acetylgalactosamine of glycosphingolipids.  相似文献   

18.
A new assay method for glycosphingolipid glycosyl-transferase activities was developed using a 96-well filtration plate of a MultiScreen assay system. An acceptor glycosphingolipid and a donor radioactive nucleotide sugar were incubated with an enzyme source in a well of the filtration plate. After incubation, both identification and quantification of the reaction product were carried out simultaneously using a specific antibody for the product which was trapped on a filtration membrane of the plate as a complex with Staphylococcus aureus protein A (IgGSorb). This assay method was used for determining the activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:Lcn4Cer alpha 2----6sialyltransferase and uridine 5'-diphosphate-N-acetyl galactosamine:GM3 N-acetylgalactosaminyltransferase. In addition to the simple and rapid identification and quantification of the product, this method proved to be as reliable and sensitive as the previously published assay procedures. Furthermore, this assay method can be used with a high concentration of detergent which should not be used in the other procedures described previously using enzyme-linked immunosorbent assay methods on a 96-well multiplate even if the enzyme reaction might require a certain percentage of the detergent concentration.  相似文献   

19.
The ELISA and GM1-ELISA, by using antiserum to purified Salmonella enterotoxin (SE), were standardized and carried out to screen salmonellae isolated from foods of animal origin for enterotoxigenicity. Of the 101 strains of Salmonella belonging to 15 different serogroups tested, 76 (75.24%) strains from 13 serogroups were found enterotoxigenic. ELISA correlated well with rabbit ligated ileal loop (RLIL) test for the detection of enterotoxin producing salmonellae with 24 test strains. ELISA also yielded positive reaction with 7 of 13 RLIL negative strains. GM1-ELISA could not be carried out as none of the 101 cell free culture supernatants (CFCS) were able to bind with GM1-ganglioside. ELISA and GM1-ELISA were also standardized with antiserum to cholera toxin for the detection of salmonellae producing cholera related enterotoxin. None of the 101 strains was found to produce cholera related enterotoxin. ELISA could detect as low as 15 ng/100 microliters of purified SE and 10 ng/100 microliters of cholera toxin when tested with their homologous antisera.  相似文献   

20.
Minke WE  Roach C  Hol WG  Verlinde CL 《Biochemistry》1999,38(18):5684-5692
Ganglioside GM1 is the natural receptor for cholera toxin (CT) and heat-labile enterotoxin (LT), which are the causative agents of cholera and traveler's diarrhea, respectively. This observation suggests that small molecules interfering with this recognition process may prevent entry of the toxins into intestinal cells, thereby averting their devastating effects. Here, the terminal sugar of ganglioside GM1, galactose, was chosen as a lead in designing such receptor antagonists. Guided by the experimentally determined binding mode of galactose, we selected a "substructure" for searching the Available Chemicals Database, which led to the purchase of 35 galactose derivatives. Initial screening of these compounds in an LT ELISA revealed that 22 of them have a higher affinity for LT than galactose itself. A structurally diverse subset of these galactose derivatives was selected for determination of IC50 values in the LT ELISA and IC50 values in a CT assay, as well as for the determination of Kd's using the intrinsic fluorescence of LT. The best receptor antagonist found in this study was m-nitrophenyl alpha-galactoside with an IC50 of 0.6 (2) mM in the LT ELISA and 0.72 (4) mM in the CT assay, 100-fold lower than both IC50 values of galactose. Careful analysis of our binding data and comparison with crystal structures led to the derivation of correlations between the structure and affinity of the galactose derivatives. These characteristics will be used in the design of a second round of LT and CT receptor antagonists.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号