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1.
β-Glucuronidase and sulfatase are the major deconjugating enzymes used in the cleavage of the glucuronate and sulfate moieties, respectively, from certain conjugated food factors including polyphenols. In the present study, we found that compounds having the same molecular weights as catechins were present in Helix pomatia- and/or Abalone entrails-derived β-glucuronidase and sulfatase by liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring methods. On the other hand, the same molecular weights as catechins were undetectable in Escherichia coli-derived β-glucuronidase and Aerobacter aerogenes-derived sulfatase. By high performance liquid chromatography, enzyme-derived catechins were not detected because of approximately 1,000-fold lower sensitivity as compared to LC-MS/MS. These results suggest that the catechins in these enzymes might be attributed to the diets of the organisms as the enzyme sources. 相似文献
2.
《Biocatalysis and Biotransformation》2013,31(5):179-185
AbstractFive commercial enzyme preparations were screened for hydrolysis of the glucuronic acid units of glycyrrhizin (GL) and baicalin. Two preparations hydrolyzing GL to glycyrrhetic acid (GA) and four enzyme preparations hydrolyzing baicalin to baicalein were obtained. One enzyme preparation with the ability to hydrolyze both GL and baicalin, namely Rapidase Pineapple, was purified by anion exchange, cation exchange and molecular sieve chromatography. The results of purification indicated that the enzymes containing the glycyrrhizin-β-d-glucuronidase (GBDG) and baicalin-β-d-glucuronidase (BBDG) activities were distinct, with different substrate specificities, molecular weights and enzymatic characteristics. GBDB hydrolyzed GL to GA, but had no detectable activity on baicalin, and BBDG hydrolyzed baicalin to baicalein, but could not hydrolyze GL. However, both GBDG and BBDG could hydrolyze the artificial substrate p-nitrophenyl- β-d-glucuronide (pNPGA). 相似文献
3.
Kotani A Miyashita N Kusu F 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,788(2):269-275
Determination of catechins in human plasma was carried out by high-performance liquid chromatography with electrochemical detection using a microbore octadecylsilica column. Peak heights for catechins were found to be linearly related to the amount of each catechin injected, from 2 pmol/ml to 2 nmol/ml (r>0.999). Conjugated-form catechins in plasma were hydrolyzed enzymatically using beta-glucuronidase and sulfatase. Catechins in plasma and the hydrolyzed solution were extracted with ethyl acetate and determined by the present method. The time courses of concentrations of catechins in human plasma showed maxima at 1-2 h after ingestion of 340 ml of commercial canned green tea. 相似文献
4.
Summary Sections of retinas from albino and pigmented rats were studied histochemically by the naphthol and lead methods for lysosomal enzymes (acid phosphatase, aryl sulfatase, glucosaminidase and -glucuronidase). Activity of the enzymes studied (except -glucuronidase) is demonstrable in granules which by their staining properties are identified as lysosomes. The matrix of the lysosome stains positively in PAS preparations and is diastaae resistant. The organelles are distributed mainly in the pigment epithelium, outer limiting membrane, inner nuclear layer, ganglion layer and inner limiting membrane of the retina.This investigation was supported by a generous grant from the Nuffield Foundation. 相似文献
5.
Suvendra Nath Bagchi Shobha Sondhia Manish Kumar Agrawal Sonali Banerjee 《Journal of applied phycology》2016,28(1):177-180
Whole cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of Anabaena fertilissima CCC597 revealed the presence of a microginin (M r?=?565 Da), which was purified using ODS C18 solid-phase extraction followed by quaternary methyl ammonium (QMA)-ion exchange and reverse-phase liquid chromatography. Its partial structure was deduced upon LC-MS/MS as β-amino-α-hydroxy-decanoic acid-Ala-MeLeu-Tyr. Microginin-565 inhibited rabbit angiotensin-converting enzyme activity at an apparent IC50?=?9.8 μM. 相似文献
6.
Asokan Rethinaswamy Chul-Hak Yang Prakash N. Srivastava 《Molecular reproduction and development》1994,38(4):404-409
Bull seminal plasma contains high levels of β-glucuronidase. The present study describes the isolation and characterization of β-glucuronidase, and its role in fertilization. β-glucuronidase was purified by ion exchange chromatography, saccharolactone-agarose affinity chromatography, and gel filtration. The specific activity of the purified enzyme was 4,414 μmoles/mg protein/min. The purified enzyme showed a single band on 7.5% PAGE. On SDS-PAGE, the enzyme appeared to consist of four identical subunits of Mr 75,000 each. The apparent Km and Vmax for β-glucuronidase were 0.4 mM and 5.7 μmol/min using phenolpthalein mono-β-glucuronic acid as the substrate. β-glucuronidase appeared to accelerate the cumulus dispersion in vitro. © 1994 Wiley-Liss, Inc. 相似文献
7.
Human polymorphonuclear leukocyte-derived enzymes, β-glucuronidase, β-N-acetylgalactosaminidase, and aryl sulfatase were studied for their ability to degrade chondroitin sulfate. Evidence is presented which implies a concerted, synergistic mechanism of action for these enzymes in glycosaminoglycan degradation excluding the possibility of endoglycosidase activity. Conclusions are based upon results derived from pH optimum, inhibitor studies, kinetics, and partial purification of these enzymes. The data presented here demonstrate that the polymorphonuclear leukocytes contain all of the enzymes necessary for the solubilization and complete degradation of the chondroitin-4-sulfate of cartilage matrix. 相似文献
8.
The β-ketoadipate pathway is a major pathway involved in the catabolism of the aromatic compounds in microbes. The recent progress in genome sequencing has led to a rapid accumulation of genes from the β-ketoadipate pathway in the available genetic database, yet the functions of these genes remain uncharacterized. In this study, the protocatechuate branch of the β-ketoadipate pathway of Rhodococcus jostii was reconstituted in vitro. Analysis of the reaction products of PcaHG, PcaB, and PcaL was achieved by high-performance liquid chromatography. These reaction products, β-ketoadipate enol-lactone, 3-carboxy-cis,cis-muconate, γ-carboxymuconolactone, muconolactone, and β-ketoadipate, were further characterized using LC-MS and nuclear magnetic resonance. In addition, the in vitro reaction of PcaL, a bidomain protein consisting of γ-carboxy-muconolactone decarboxylase and β-ketoadipate enol-lactone hydrolase activities, was demonstrated for the first time. This work provides a basis for analyzing the catalytic properties of enzymes involved in the growing number of β-ketoadipate pathways deposited in the genetic database. 相似文献
9.
U Gräfe S Heinze B Schlegel A Härtl 《Journal of industrial microbiology & biotechnology》2001,27(3):136-143
The application of modern mass spectrometry methods (SI-CID-MS/MS; MS
n
) in the disclosure of new and recurrent microbial metabolites is discussed. Spray ion (SI) sources coupled to different kinds
of mass analyzers enable the determination of molecular weights and chemical formulas of given samples even in mixtures. Diagnostic
fragment formation by collision-induced dissociation (CID-MS/MS) and MS
n
experiments using ion trap mass analyzers are shown as another indispensable source of structural information. Due to the
development of benchtop-type mass spectrometers coupled to high-performance liquid chromatography (HPLC), MS can be practised
in almost every laboratory as a powerful tool in natural product analysis. Examples are given for special MS applications
in identification of bioactive metabolites from screening strains. Journal of Industrial Microbiology & Biotechnology (2001) 27, 136–143.
Received 21 September 1999/ Accepted in revised form 19 January 2000 相似文献
10.
Fast protein liquid chromatography on Superose 6 of crude extracts from the green alga Chlorella kessleri cultivated autotrophically in white light reveals several peaks with phosphofructokinase (PFK, EC 2.7.1.11) or pyruvate kinase (PK, EC 2.7.1.40) activity with molecular weights larger than the usually reported ones of 320–380 and 240 kDa, respectively. All other glycolytic enzymes are eluted as one peak each with a molecular weight corresponding to data from the literature. Indirect evidence indicates that the various forms of PFK and presumably PK are oligomers. The occurrence of different PFK species depends markedly on growth conditions such as wavelength of light: Red light leads to only one rather large PFK (1,580 kDa), blue light to two smaller species (760 and 360 kDa). All species are probably present in white light-grown cells (1,500, 1,050, 930, 700 and 440 kDa). The various light qualities do not significantly affect all other glycolytic enzymes. PK constantly exhibits four forms with molecular weights of 830, 680, 480, 305 kDa. Experiments with the chlorophyll-free mutant no. 20 of Chlorella kessleri support the assumption that oligomerization of enzymes is characteristic of regulatory enzymes, thereby providing the cell with an additional regulatory means. 相似文献
11.
采用硅胶柱层析及制备型液相色谱仪对红曲米中两种荧光物质进行分离纯化,使用高效液相色谱法(HPLC)检测荧光物质纯度,然后使用高分辨质谱(ESI-HRMS)对两种荧光物质进行分析,得到两种荧光物质的分子量分别为356和384,ESI-MS/MS二级质谱把两者鉴定为monasfluore A (MFA)和monasfluore B (MFB);从金华地区红曲米中分离得到10株红曲菌株,经固态发酵采用HPLC法分析,筛选获得1株高产MFA、MFB的菌株WZWZ,该菌株发酵制得红曲米中MFA含量为3.63 g/kg,MFB含量为7.29 g/kg,对WZWZ菌株进行外观形态学及显微观察、ITS基因序列测定与分析,最终将菌株WZWZ鉴定为紫色红曲霉(Monascus purpureus)。 相似文献
12.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,751(1):93-105
A new method for a comprehensive screening and confirmation of β-2 agonists in human urine is presented based on gas chromatography–low-resolution mass spectrometry (GC–MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with β-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other β-2 agonists remain unchanged. Liquid–liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four β-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other β-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented. 相似文献
13.
Mass spectrometric profiling using ProteinChip and magnetic beads has rapidly grown over the past years, particularly to generate serum profiles for cancer diagnosis. The molecular weights of these distinguishing peaks are usually under 30 kDa. To identify those low molecular weight proteins and peptides is important for specific assays to be developed and increases biological insight. In this study, low molecular weight proteins and peptides from serum were purified by a combination of weak cation exchange magnetic beads and high performance liquid chromatography. The purified proteins and peptides were analyzed by 1D SDS PAGE, SELDI and LC-MS/MS. 246 proteins were identified from the HPLC fractions by LC-MS/MS. 95(38.62%) proteins were first identified in serum compare with Sys-BodyFluid database. 11(11/96) proteins were documented cancer associated proteins. We also observed about 109 proteins/peptides in SELDI mass spectrum, and 13 of the SELDI features were identified. 相似文献
14.
Clearance experiments with highly purified lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, following intravenous infusion revealed widely varying clearance profiles which depended on the tissue source of the enzyme. Normal rat serum β-glucuronidase and epididymal N-acetyl-β-d-glucosaminidase were cleared slowly from the circulation when compared with rat preputial gland β-glucuronidase, liver lysosomal β-glucuronidase, and liver lysosomal N-acetyl-β-d-glucosaminidase, respectively, which were cleared rapidly. Experiments comparing the catalytic properties and molecular dimensions of the enzymes revealed no differences between rapid and slow clearance forms. Kinetic analysis using the rapid clearance forms of β-glucuronidase has allowed the resolution of at least two components, rapid and slow. Clearance of the rapid component is saturable and appears to reflect binding or uptake by a limited number of sites. By contrast, the clearance rate of the slow component increased linearly with respect to dose and may be due to nonspecific or low-affinity binding. Competition experiments with β-glucuronidase-free lysosomal extract and highly purified lysosomal enzymes, but not serum glycoproteins or colloidal silver, suggest that one lysosomal enzyme inhibits clearance of others and that a common mechanism may be involved in their binding. 相似文献
15.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,754(1):229-244
A liquid chromatography–mass spectrometry (LC–MS) method for the analysis of corticosteroids in equine urine was developed. Corticosteroid conjugates were hydrolysed with β-glucuronidase; free and enzyme-released corticosteroids were then extracted from the samples with ethyl acetate followed by a base wash. The isolated corticosteroids were detected by LC–MS and confirmed by LC–MS–MS in the positive atmospheric pressure chemical ionisation mode. Twenty-three corticosteroids (comprising hydrocortisone, deoxycorticosterone and 21 synthetic corticosteroids), each at 5 ng/ml in urine, could easily be analysed in 10 min. 相似文献
16.
Jeevan K. Prasain Ning Peng Yanying Dai Ray Moore Alireza Arabshahi Landon Wilson Stephen Barnes J. Michael Wyss Helen Kim Ray L. Watts 《Phytomedicine》2009,16(2-3):233-243
Proanthocyanidin rich plant extracts derived from grape seed extract (GSE), hawthorn and cranberry are on markets for their preventive effects against cardiovascular diseases and uroinfections in woman. However, the importance of these health beneficial effects of these botanicals remains elusive due to incomplete understanding of uptake, metabolism and bioavailability of proanthocyanidins in vivo. In the present study rats were given GSE orally (300 mg/kg, twice a day) and blood and urine were collected over a 24 h period. Monomeric catechins and their methylated metabolites, and proanthocyanidins up to trimers were detected in blood samples treated with GSE using LC-MS/MS operating in the multiple reaction monitoring (MRM) mode. A new tetramethylated metabolite of dimeric proanthocyanidin (m/z 633) in GSE-treated urine was tentatively identified. Using LC-MS/MS, (+)-catechin and (?)-epicatechin were identified in the brain conclusively. These data suggested that GSE catechins cross the blood brain barrier and may be responsible for the neuroprotective effects of GSE. 相似文献
17.
Ramírez-Mata A Michalak C Mendoza-Hernández G León-Del-Río A González-Noriega A 《Experimental cell research》2011,(16):2364-2373
Endocytosis and transport of bovine liver β-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI–MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine β-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine β-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine β-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine β-glucuronidase. 相似文献
18.
Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) after hydrolysis with β-glucuronidase and liquid–liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of metandienone metabolism by LC–MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC–MS/MS and LC–MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction). 相似文献
19.
P Schlesinger J S Rodman M Frey S Lang P Stahl 《Archives of biochemistry and biophysics》1976,177(2):606-614
Certain highly purified forms of rat lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, are rapidly cleared from the circulation following intravenous infusion. Several lines of evidence are presented which indicate that the primary site of enzyme uptake is the liver. Clearance of the two enzymes was unaffected by nephrectomy, whereas it was abolished by evisceration. Tissue distribution experiments with native and [125I]β-glucuronidase indicate the liver as the major, if not exclusive, site of enzyme uptake. Experiments with the isolated perfused liver showed clearance of certain enzyme preparations but not others. Those enzymes cleared by the isolated perfused liver were likewise cleared in vivo. Liver fractionation studies following infusion of large doses of β-glucuronidase revealed a rapid, short-lived increase in microsomal β-glucuronidase and a slower but larger increase in lysosomal β-glucuronidase. The results indicate that β-glucuronidase, N-acetyl-β-d-glucosaminidase, and probably other glycosidases are rapidly incorporated into the lysosomal compartment of liver. 相似文献
20.
Fernandes RM Gomes GC Porcari AM Pimentel JR Porciúncula PM Martins-Júnior HA Miguez PH da Costa JL Amaral PH Perecin F Meurer EC Furtado PV Simas RC Eberlin MN Ferreira CR Madureira EH 《Theriogenology》2011,76(7):1266-1274
Quantitation of progesterone (P4) in biological fluids is often performed by radioimmunoassay (RIA), whereas liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been used much less often. Due to its autoconfirmatory nature, LC-MS/MS greatly minimizes false positives and interference. Herein we report and compare with RIA an optimized LC-MS/MS method for rapid, efficient, and cost-effective quantitation of P4 in plasma of cattle with no sample derivatization. The quantitation of plasma P4 released from three nonbiodegradable, commercial, intravaginal P4-releasing devices (IPRD) over 192 h in six ovariectomized cows was compared in a pairwise study as a test case. Both techniques showed similar P4 kinetics (P > 0.05) whereas results of P4 quantitation by RIA were consistently higher compared with LC-MS/MS (P < 0.05) due to interference and matrix effects. The LC-MS/MS method was validated according to the recommended analytical standards and displayed P4 limits of detection (LOD) and quantitation (LOQ) of 0.08 and a 0.25 ng/mL, respectively. The high selective LC-MS/MS method proposed herein for P4 quantitation eliminates the risks associated with radioactive handling; it also requires no sample derivatization, which is a common requirement for LC-MS/MS quantitation of steroid hormones. Its application to multisteroid assays is also viable, and it is envisaged that it may provide a gold standard technique for hormone quantitation in animal reproductive science studies. 相似文献