Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.
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PMID:Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster. 10 85

An alpha-glucosidase was purified from baker's yeast. The molecular weight was approximately 44 000 daltons. SDS-disc gel electrophoresis suggested that the enzyme consisted of four subunits. The isoelectric point was at pH 5.4. The Km values for p-nitrophenyl alpha-D-glucopyranoside and maltose were 2.9 X 10(-4) and 2.5 X 10(-2) M, respectively. Binding of 2-(p-toluidino)naphthalene-6-sulfonate to the alpha-glucosidase was associated with a strong increase in fluorescence. The dissociation constant of the enzyme-TNS complex was 8 X 10(-5) M. The fluorescent probe did not interfere with the binding of glucose to the enzyme although the alpha-glucosidase was inhibited by high concentrations of TNS. The formation of an enzyme-glucose complex was indicated by an increase of fluorescence and by a shift in the wavelength for maximal emission which suggests that the binding process is associated with a change in conformation. The dissociation constant of the glucose--alpha-glucosidase complex KD = 0.57 X 10(-3) M, was calculated from the increase in fluorescence as a function of glucose concentration.
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PMID:On the properties of alpha-glucosidase and the binding of glucose to the enzyme. 36 Jul 36

Turnover in organ culture of human small intestinal membrane glycoproteins was measured by the pulse-chase technique, using 14C-glucosamine, 14C-fucose or 14C-leucine as tracers. Apparently, low degradation rates were found for the major high-molecular-weight proteins which co-migrated on SDS-polyacrylamide gels with maltase-glucoamylase, lactase-phlorizin-hydrolase and sucrase-isomaltase enzymic activities. In contrast, an unidentified glycoprotein appearing on gels next to alkaline phosphatase exhibited a higher degradation rate with an apparent half-life of about 30 h, this being similar to the half-life of total glycoprotein as measured in mucosal homogenates. The results obtained with the pulse-chase technique were confirmed by double isotope experiments using 14C-leucine and 3H-leucine as tracers. These findings indicate that in organ culture there is a low basic turnover of human intestinal membrane glycoproteins which co-migrate on gels with known glycosidase enzymic activities.
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PMID:Turnover studies of human intestinal brush border membrane glycoproteins in organ culture. 45 41

Sucrase-isomaltase complex and its functional subunits have been identified in homogenates of human small intestinal mucosa by use of Sephadex G-200 (superfine) chromatography aided by affinity of the isomaltase moiety for the dextran gel. The isomaltase subunit binds strongly to the gel at 4 degrees, and is eluted only after 2 column volumes; earlier recovery as a sharp peak can be achieved by raising column temperature to 37 degrees after elution of other proteins. Bio-Gel P-300 chromatography, density gradient, and equilibrium centrifugation demonstrated that the sucrase subunit (Stokes radius = 45 A, frictional ratio = 1.32, s20,w = 6.9, MW = 130,000) and the isomaltase subunit (Stokes radius = 45 A, frictional ratio = 1.30, s20,w = 6.6, MW = 120,000) are similar but unequal in size. The sucrase-isomaltase complex (Stokes radius = 70 A, frictional ratio = 1.61, s20,w = 9.8, MW = 280,000), appears to be an elongated hybrid molecule that is less symmetrical than either of itt subunits. Apparent Km and pH activity curves were indistinguishable for each enzyme whether present in the hybrid or in the free state. The sucrase-isomaltase complex, accounting for approximately 90 percent of native intestinal sucrase and isomaltase activities, was isolated and cleaved by 0.01 M beta-mercaptoethanol/6 M urea treatment into active sucrase and isomaltase subunits having biochemical characteristics identical with those of the free native moieties. Sodium dodecyl sulfate acrylamide gell electrophoresis of the complex also produced subunits having molecular weights very close to those for the active free sucrase and isomaltase moieties, indicating that each alpha-glucosidase appears to consist of a single polypeptide chain. Immunization of rabbits with pure sucrase-isomaltase complex yielded a monospecific precipitating antibody that reacted with the hybrid and the sucrase subunit, but had minimal affinity for the isomaltase subunit, providing further evidence that the sucrase-isomaltase molecule is a hybrid consisting of two distinct alpha-glucosidases.
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PMID:Human intestinal sucrase-isomaltase. Identification of free sucrase and isomaltase and cleavage of the hybrid into active distinct subunits. 80 75

A highly thermostable alpha-glucosidase (E C.3.2.1.20) from an extreme thermophile, Thermus thermophilus HB 8, was purified to homogeneous by ammonium sulfate fractionation, DEAE-cellulose chromatography and preparative slab gel electrophoresis. The enzyme was purified 17 fold with 21% recovery of activity. The enzyme had a molecular weight of 67000 by SDS-PAGE. The isoelectric point was pH4.5 by IEF on PAG. The enzyme hydrolized p-nitrophenyl-alpha-glucoside (PN-PG), sucrose and maltose, but not cellobiose, melibiose and soluble starch. The km value for PNPG was 0.4mmol/L, the Vmax was 0.29 mumol.min-1.mg-1. The enzyme exhibited high thermostability. After incubation at 90 degrees C for 10 h in 50 mmol/L acetate buffer pH 5.8, the enzyme retained 90% of its original activity. The half-live (t1/2) at 95 degrees C was 108 min. The enzyme was activated by Mg2+, Mn2+, Ca2+, Ba2+ and strongly inhibited by Hg2+, Cu2+. Modification of the enzyme by EDC or DEPC led to complete loss of activity, which suggests that carboxyl group(s) and histidine residue(s) are essential for activity of alpha-glucosidase.
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PMID:[Purification and characterization of alpha-glucosidase from an extreme thermophile, Thermus thermophilus HB 8]. 141 35

The common hookworm (Ancylostoma ceylanicum) infection of humans was studied in golden hamsters model system. Significant biochemical modulations were observed in hamster jejunal brush border membrane (BBM), the primary site of infection. Analysis of BBM at the peak of infection (3-weeks) revealed a marked decrease in the activities of sucrase, lactase and maltase, while activities of alkaline phosphatase, (Ca2+ + Mg2+)-ATPase and gamma-glutamyl transpeptidase were increased. Kinetic studies conducted with maltase, a superficially localised enzyme of jejunal BBM, revealed loss of enzyme active site during the infection. Among other constituents, the levels of cholesterol and triglycerides were significantly decreased with slight increase in phospholipid content in the infected animals. The hookworm infection also caused a decline in total hexose content indicating an altered membrane glycocalyx. Conversely, there was significant enhancement of hydroxyproline and sialic acid contents. SDS-PAGE analysis showed an enhancement in both low and high molecular weight proteins in jejunal BBM preparations of the infected group. Gel electrophoresis of glycoproteins further revealed the appearance of two additional peaks in the low molecular weight region and concomitant disappearance of a peak in the high molecular weight region. These results strongly support the view that the hookworm infection causes severe damage not to the site of attachment alone but also to the entire cell lining of the jejunum and therefore could influence overall digestion and absorption.
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PMID:Biochemical analysis of jejunal brush border membrane of golden hamster: pathogenic modulations due to ancylostomiasis. 159 19

To clarify the enzyme participates in maltotriose synthesis, we purified maltase from rabbit kidney using 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) affinity column chromatography. The purified enzyme possessed specific activity of 33.7 mumol/mg/min and estimated molecular weight of 350,000 dalton, as judged by SDS-polyacrylamide gel electrophoresis, comparable with those reported from rat kidney. Moreover this enzyme possessed not only maltase (maltose----glucose) but also amylomaltase (maltose----maltotriose) activity, and both activities were inhibited by Tris in a dose-dependent manner with similar IC50 values. From these results, we concluded that maltotriose was synthesized by maltase in vitro and that kidney maltase may participate in sugar metabolism in vivo.
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PMID:Synthesis of maltotriose by maltase purified from rabbit kidney. 177 60

Highly active glycoprotein allergens have been isolated from pollen of Prosopis juliflora by a combination of Sephadex G-100 gel filtration and Sodium dodecyl sulphate-Poly-acrylamide gel electrophoresis. The glycoprotein fraction was homogeneous, and had molecular weight 20,000. The purified glycoprotein allergen contained 20% carbohydrate, mainly arabinose and galactose. Enzymatic digestion of glycoprotein with protease released glycopeptides of molecular weight ranging from less than 1,000 to more than 5,000 on Sephadex G-25 gel filtration. Antigenicity or allergenicity testing of these glycopeptides by immunodiffusion, immunoelectrophoresis, and radioallergosorbent test indicated complete loss of allergenic activity after digestion with protease whereas incubation with beta-D-galactosidase and periodate oxidation had little affect on the allergenic activity of the glycoprotein fraction. But incubation with alpha-D-glucosidase did not affect the allergenic activity significantly. All these tests indicated that protein played significant role in allergenicity of P. juliflora pollen.
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PMID:Purification and characterization of the glycoprotein allergen from Prosopis juliflora pollen. 187 83

Intestinal brush borders from Wistar rats contained a total of 20-30-times more binding sites for Escherichia coli heat-labile enterotoxin (LT-1) than for cholera toxin (CT). The results suggest that LT-1 binds to sites in addition to ganglioside GM1, the binding site for CT. Brush border proteins were separated by SDS-PAGE, blotted to nitrocellulose and the filters incubated with 125I-labeled toxins. [125I]LT-1 was shown to bind to a series of brush border galactoproteins ranging in size from 130-140 kDa. Binding was inhibited by unlabeled LT-1 (but not CT), and by ricin and free galactose. A number of brush border enzymes are large glycoproteins which can be solubilised by papain. The papain-solubilised sucrase-isomaltase complex was purified by affinity chromatography and shown to bind LT-1, as did the proteins in fractions enriched in maltase activity. However, such brush border galactoproteins do not account for all of the additional LT-1 binding sites. Thus, brush borders prepared from 1-15-day-old rabbits contained many more binding sites for LT-1 than CT despite the absence of any sucrase-isomaltase activity, and no [125I]LT-1 binding proteins could be detected by blotting. There was a marked variation in the number of LT-1 binding sites in different strains of rat, and between different species.
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PMID:Characterisation of the binding sites for Escherichia coli heat-labile toxin type I in intestinal brush borders. 193 71

Acid alpha-glucosidase purified from pig liver showed heterogeneity in its affinity to Sephacryl S-200 gel. Acid alpha-glucosidase was separated into two fractions (S1 and S2) by Sephacryl S-200 affinity chromatography. Each fraction contained components at apparent 76 kDa and 67 kDa on SDS/PAGE. The amount of S1 fraction was about 1.3 times that of the S2 fraction. In the kidney the ratio of S1 to S2 fraction was similar to that in the liver. However, the heart contained 1.3 times as much S2 fraction as S1 fraction. The spleen acid alpha-glucosidase consisted mainly of S1 fraction, containing only a 76 kDa component. Immunohistochemically, acid alpha-glucosidase was demonstrated in the macrophages of the spleen. Thus the 76 kDa component in the spleen must come mainly from the macrophages. Lectin-binding analysis was carried out on the components present in the S1 and S2 fractions after electrophoresis and transfer to nitrocellulose sheets. Slight differences in binding observed suggest differences in the structure of the sugar side chains.
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PMID:Heterogeneity of pig lysosomal acid alpha-glucosidase. Affinity to Sephacryl S-200 gel and tissue distribution. 195 64


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