Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.20 (alpha-glucosidase)
 4,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We described a partitioned enzyme-sensor system, which incorporates an immoblized substrate and three or more discrete immobilized enzymes. This instrument measures alpha-amylase activity by passing the solution containing alpha-amylase over a column packed with immobilized starch. The resulting oligosaccharides are successively exposed to a column or columns containing immobolized glucose oxidase, catalase, glucoamylase or maltase, and glucose oxidase. The resulting hydrogen peroxide is detected by a three-electrode amperometric cell. All immobilized reagents were immobilized on a particulate, porous alumina to allow rapid and constant flow rate. With use of less than optimum immobilized reagents, alpha-amylase activity has been measured from about 5 to 200 kU/liter with a 50 microliter sample size. Lack of sensitivity is predominantly attributable to the low activity and low stability of immobilized maltase and glucoamylase. We believe that a clinical test using this system is feasible and desirable because the immobilized reagent system should allow for testing of alpha-amylase with excellent precision, convenience to the operator, and low cost.
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PMID:Coupled reactions of immobilized enzymes and immobilized substrates: clinical application as exemplified by amylase assay. 35 22

Two methods for specifically detecting maltase, alpha-glucosidase, or isomaltase activity in electrophoresis gels are described. Both systems couple the formation of glucose by enzyme action on maltose or isomaltose to the generation of a colored product. System A uses an agarose overlay which contains substrate, glucose oxidase, peroxidase, 2,4-dichlorophenol, and 4-L-amino-phenazone. A purple color is produced at the site of enzyme activity. No hazardous chemicals are used at any stage. The stain is simple, rapid, sensitive, and inexpensive and does not interfere with subsequent protein staining. However, the stain is not permanent. System B was developed to give a permanent stain. The gel is overlaid with agarose containing substrate, glucose oxidase, phenazine methosulfate, and nitroblue tetrazolium. Glucose production results in the nitroblue tetrazolium being oxidized to an insoluble formazan with a dark blue color. This stain is also sensitive, rapid, and inexpensive but does use hazardous chemicals and if overstaining occurs this can interfere with subsequent protein staining. Neither system inactivates the localized enzymes which can be recovered from the gel if desired.
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PMID:Two staining methods for selectively detecting isomaltase and maltase activity in electrophoresis gels. 169 32

The enzymic hydrolysis of glycosyl fluorides is conveniently followed by using a pH-stat. Reactions involving glucosyl or galactosyl fluorides can also be followed by using glucose oxidase or galactose oxidase respectively. The pH-stat allows the rapid assay of intestinal alpha-glucosidase in crude homogenates. Use of glycosyl fluorides as substrates for glycosidases facilitates the polarimetric or g.l.c. determination of the anomeric nature of the initial product of hydrolysis. Hydrolysis by fungal amyloglucosidase proceeds with inversion of configuration whereas that by yeast and rat intestinal alpha-glucosidase, coffee-bean alpha-galactosidase and almond emulsin beta-glucosidase proceeds with retention of configuration. beta-d-Glucopyranosyl azide was not a detectable substrate for almond emulsin beta-d-glucosidase.
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PMID:The hydrolysis of glycosyl fluorides by glycosidases. Determination of the anomeric configuration of the products of glycosidase action. 512 11

Maltase from Saccharomyces cerevisiae-II was purified by ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing. The purification procedure resulted in two enzyme isoforms with pI of 5.35 and 5.3 and identical specific activities. The molecular weights of the isoforms as determined by SDS polyacrylamide gel electrophoresis and gel filtration through Sephadex G-100 are 60 000 and 55 000, respectively. Both isoforms were electrophoretically polydisperse. The maltase isoforms are glycoproteins containing 1.5-2% of glucosamine and 5-8% (isoform A) and 2-3% (isoform B) of neutral sugars. Using paper chromatography and glucose oxidase, it was shown that glucose is an indispensable constituent of neutral sugars in both isoforms.
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PMID:[Isolation and properties of maltase from Saccharomyces cerevisiae-II]. 701 1

We discuss, from an industrial point of view, the scope and possibilities of recombinant DNA technology for "diagnostic enzyme" production and application. We describe the construction of enzyme-overproducing strains and show how to simplify downstream processing, increase product quality and process profitability, improve diagnostic enzyme properties, and adjust enzymes to harsh assay conditions. We also consider some safety and environmental aspects of enzyme production. Other aspects of diagnostic enzymes that we cover are the facilitation of enzyme purification by attachment of short amino acid tails, the introduction of tails or tags for site-specific conjugation or oriented immobilization, the construction of bi- or multifunctional enzymes, and the production of enzyme-based diagnostic tests as demonstrated by the homogeneous immunoassay system of CEDIA tests. We use as examples of diagnostic enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucose oxidase (EC 1.1.3.4), alkaline phosphatase (EC 3.1.3.1), alpha-glucosidase (EC 3.2.1.20), pyruvate oxidase (EC 1.2.3.3), creatinase (EC 3.5.3.3), and beta-galactosidase (EC 3.2.1.23).
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PMID:Enzymes in diagnostics: achievements and possibilities of recombinant DNA technology. 817 39

If a fermentation process is followed by a change in the concentration of maltose, information on the level of the maltose is important. A new type of sensor was developed for measuring the content of maltose in fermentation broth. The base of the sensor is a thin-layer reactor, in which a protein membrane was chosen for the immobilization of enzymes. To measure maltose, we investigated the influence of enzymes such as alpha-glucosidase (EC 3.2.1.20) and amyloglucosidase (EC 3.2.1.3) on the effectiveness of conversion to glucose. Because amyloglucosidase proved to be more effective, a mixture of amyloglucosidase and glucose oxidase was applied for the determination of maltose. To develop the best measuring technique, the consequences of changes in different parameters, such as the optimal ratio of enzymes, role of pH value and that of the flow rate, were studied. An amperometric measuring cell with Pt-Ag/AgCl-Pt electrodes was used at +600 mV operating potential. The results indicate that the maltose content in different types of fermentation broth can be determined by the new measuring cell in the range of 0.2-4 mM maltose. The cell was successfully tested in the fermentation of brewer's yeast.
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PMID:Studying the bienzyme reaction with amperometric detection for measuring maltose. 825 Nov 37

This study describes a rapid, sensitive, and automated spectrophotometric enzymatic microassay that measures the intracellular glycogen of primary cultured hepatocytes and other cultured cells in 96-well plates and can be adapted for other samples that are transferred to these plates. The procedure involves in situ disruption of cells, followed by hydrolysis of glycogen into glucosyl units by fungal glucoamylase (exo-1.4-alpha-glucosidase, EC 3.2.1.3), and glucose determination with the glucose oxidase colorimetric method. The color intensity can be measured in conventional ELISA readers, and the data can be fed to an on-line computer for rapid processing. The advantages of this method are its simplicity and automation, the reduction in sample handling, and the small number of cells required compared to other conventional methods.
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PMID:A microassay for measuring glycogen in 96-well-cultured cells. 866 May 8

Micrometer-sized enzyme grids were fabricated on gold surfaces using a novel method based on a flow-through microdispenser. The method involves dispensing very small droplets of enzyme solution (approximately 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzle of a microdispenser, resulting in enzyme patterns with a line width of approximately 100 microm. Different immobilization methods have been evaluated, yielding either enzyme monolayers using functionalized self-assembled thiol monolayers for covalent binding of the enzyme or enzyme multilayers by cross-linking or entrapping the enzymes in a polymer film. The latter immobilization techniques allow the formation of coupled multienzyme structures. On the basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha-glucosidase, mutarotase, and glucose oxidase) microstructures consisting of line patterns of one enzyme intersecting with the patterned lines of the other enzyme(s) were fabricated. By means of scanning electrochemical microscopy (SECM) operated in the generator-collector mode, the enzyme microstructures and their integrity were visualized using the localized detection of enzymatically produced/consumed H2O2. A calibration curve for glucose could be obtained by subsequent SECM line scans over a glucose oxidase microstructure for increasing glucose concentrations, demonstrating the possibility of obtaining localized quantitative data from the prepared microstructures. Possible applications of these enzyme microstructures for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.
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PMID:A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser. 1156 17

The quantification of glucose by using a multi-channel dissolved oxygen (DO) meter (DOX96) with immobilized glucose oxidase (GOD) and mutarotase (MUT) was performed. An evaluation of the inhibitory activities for alpha-glucosidase (AGH) by modifying our batch-type pseudo-in vivo assay system [Oki et al.; Biol Pharm. Bull., 2000, 232, 1084] was also performed using a DOX96. When 45 U/well GOD and 18.75 U/well MUT were immobilized on the surface of a gelatin membrane on the electrodes, the response shown by the decrease percent of DO (%) obtained with 8 electrode wells in the same row was linear with the glucose concentration up to 3.3 mM and a correlation coefficient larger than 0.9. To estimate the AGH inhibitory activity, AGH-immobilized Sepharose supports in the well of a silent screen plate were used. The IC50 values of acarbose and 1-deoxynojirimycin, a medicinal inhibitor for diabetes, were 0.70 +/- 0.08 microM and 0.40 +/- 0.13 microM, respectively, and coincided well with those by a pseudo-in vivo assay.
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PMID:A novel method for the assay of alpha-glucosidase inhibitory activity using a multi-channel oxygen sensor. 1250 81

An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts > 500 bp, were randomly selected and unidirectional sequenced. Forty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1 sequence showed 93% and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of alpha-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks A1, A2, B1, and C1 were 115, 55, 50, and 300 kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 : A2 : B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJP1 of A. mellifera.
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PMID:Isolation and characterization of major royal jelly cDNAs and proteins of the honey bee (Apis cerana). 1465 76


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