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)

Alpha-glucosidase III, which was different in substrate specificity from honeybee alpha-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of alpha-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in alpha-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl alpha-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of alpha-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl alpha-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai's) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.
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PMID:Purification and substrate specificity of honeybee, Apis mellifera L., alpha-glucosidase III. 1151 46

1-Deoxynojirimycin (DNM) is a saccharide decoy that inhibits cellular alpha-glucosidase I-II activity. Treatment by DNM of human immunodeficiency virus (HIV)-infected lymphocyte cultures inhibits virus spread. The functional properties of the membrane-associated Env glycoprotein (Env) modified in the presence of DNM remain unclear because previous reports on this subject have essentially used recombinant soluble Envs whose properties differ notably from those of Env anchored on the surface of the virus. To model virus-associated Env synthesized in the presence of DNM, native Env was expressed at the surface of mammalian cells treated with DNM. As expected, its glycosylation pattern was altered in the presence of the inhibitor. Env was found able to bind CD4, whereas its ability to induce membrane fusion was abolished. The immunoreactivity of regions involved in interactions of Env with CXCR4 (V1, V2, C2, and V3) was modified and Env displayed altered interaction with this coreceptor. These results are consistent with the inhibition by DNM of virus entry at the Env/coreceptor interaction step. Finally, preliminary data indicate that suboptimal concentrations of DNM and natural or synthetic CXCR4 ligands used in combination potently inhibit the Env-mediated membrane fusion process. Altogether, our results suggest that DNM and its analogs deserve further investigation as anti-HIV agents in combination with experimental compounds targeting CXCR4 to inhibit each partner of this crucial step of HIV entry.
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PMID:The alpha-glucosidase inhibitor 1-deoxynojirimycin blocks human immunodeficiency virus envelope glycoprotein-mediated membrane fusion at the CXCR4 binding step. 1175 20

The present study examines the coprecipitation of measles virus (MV) glycoproteins with host cell endoplasmic reticulum (ER) chaperone proteins. Both the haemagglutinin (H) and fusion (F) glycoproteins interacted with calnexin and GRP78, whereas interaction with calreticulin was only demonstrated for the H glycoprotein. The alpha-glucosidase inhibitor castanospermine reduced and delayed the association of F proteins with calnexin. We have previously shown that alpha-glucosidase activity is important for the functionality and antigenicity of the MV F glycoprotein and for release of MV particles from infected cells. Thus, interaction with calnexin appears vital for processing of nascent MV F protein into its functional conformation. In contrast to many other viral glycoproteins, a substantial proportion of the pulsed MV glycoproteins remained associated with ER chaperones for more than 2(1/2) h. Thus, the slow and incomplete migration of MV glycoproteins to the cell surface may result from their retention by ER chaperones, probably due to malfolding. MV infection upregulated the cellular expression of calreticulin and GRP78 and also increased their presence at the cell surface. The chaperone proteins are involved in a wide range of cellular processes, and their induction by MV may play a role for the pathogenesis of measles and its sequelae.
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PMID:The measles virus (MV) glycoproteins interact with cellular chaperones in the endoplasmic reticulum and MV infection upregulates chaperone expression. 1176 11

We have reductively alkylated deoxynojirimycin imino sugars using sodium cyanoborohydride to provide an efficient means of generating a series of N-alkylated compounds containing 4-18 carbon side chains. The yields were greater than 90% using a variety of aldehydes of different chain lengths, and after purification were >95% pure using (1)H-NMR. Radiolabelled compounds were prepared using sodium cyanoborotriti-ide to selectively label the first carbon atom in the alkyl chain and used in protein-binding and cell- and tissue-uptake experiments. Protein binding was chain-length-dependent with compounds of intermediate chain length (C(9)-C(12)), demonstrating an equal distribution between the aqueous and protein-bound phase. The extent of cell uptake also increased proportionally with increased chain length in a time-dependent manner. When administered to mice, the longer alkyl-chain compounds showed reduced absorption from the intestine and a marked deposition of compound in the liver and brain, suggesting that the more hydrophobic compounds were poorly cleared by the major tissues. In tissue-culture cells compounds with 8 or fewer carbon atoms were non-toxic and had CC(50) (the concentration at which the number of cells or cell proliferation is reduced by 50%) values greater than 1 mM. Compounds with chain lengths above C(8) showed a chain-length-dependent increase in cytotoxicity. N-alkylated deoxynojirimycins (C(4)-C(18)) were evaluated for their inhibitory effects on ceramide-specific glucosyltransferase and glycoprotein-processing alpha-glucosidase. Increasing the alkyl chain length had little effect on alpha-glucosidase activity, but inhibition of ceramide-specific glucosyltransferase increased 10-fold when C(4) and C(9)-C(18) compounds were compared. Overall these data provide further definition of the molecular features of alkylated imino sugars that influence tissue selectivity and efficacy for cellular enzyme inhibition.
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PMID:Preparation, biochemical characterization and biological properties of radiolabelled N-alkylated deoxynojirimycins. 1198 84

A soluble alpha-glucosidase presumably involved in the general carbohydrate metabolism was purified from E. histolytica trophozoites by a three-step procedure consisting of ion exchange, size exclusion and adsorption chromatographies in columns of Mono Q, Sepharose CL-6B and hydroxyapatite, respectively. After the last step, the enzyme was enriched about 673-fold over the starting material with a yield of 18%. SDS-PAGE revealed the presence in the purified preparations of two polypeptides of comparable intensity exhibiting molecular weights of 43 and 68 kDa. These results and the molecular weight of 243 kDa determined by gel filtration, suggest that the native enzyme is a heterotetramer consisting of two copies of each subunit. Some properties were investigated to determine the role of this activity in glycoprotein processing. Analysis of linkage specificity using a number of substrates indicated a preferential hydrolysis of isomaltose (alpha1,6) with much less activity on nigerose (alpha1,3) and maltose (alpha1,4). Trehalose (alpha1,1), kojibiose (alpha1,2) and cellobiose (beta1,4) were not cleaved at all. As expected, isomaltose competed away hydrolysis of 4-methylumbelliferyl-alpha-D-glucoside with a higher efficiency than nigerose and maltose. Hydrolysis of the fluorogenic substrate was competitively inhibited by glucose and 6-deoxy-D-glucose with comparable K(i) values of 0.23 and 0.22 mM, respectively. Sensitivity of the enzyme to the alpha-glucosidase inhibitors 1-deoxynojirimycin, castanospermine and australine largely depended on the substrate utilized to determine activity. 1-Deoxynojirimycin and castanospermine inhibited isomaltose hydrolysis in a competitive manner with K(i) values of 1.2 and 1.5 muM, respectively. The properties of the purified enzyme are consistent with a general glycosidase probably involved in glycogen metabolism.
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PMID:Purification and biochemical characterization of a soluble alpha-glucosidase from the parasite Entamoeba histolytica. 1457 11

Processing alpha-glucosidase I, which is encoded by CWH41, regulates one of the key steps in asparagine-linked glycoprotein biosynthesis by cleaving the terminal alpha-1,2-linked glucose from Glc(3)Man(9)GlcNAc(2), the common oligosaccharide precursor. This cleavage is essential for further processing of the oligosaccharide to the complex, hybrid, and high mannose type carbohydrate structures found in eukaryotes. A method is described for the purification of the soluble form of the alpha-glucosidase I, from recombinant Saccharomyces cerevisiae overexpressing CWH41. A homogeneous enzyme preparation was obtained in higher yield than previously reported. Cultivation of recombinant S. cerevisiae in a fermenter increased the biomass 1.7 times per liter and enzyme production 2 times per liter compared to cultivation in shake flasks. Ammonium sulfate precipitation with three chromatography steps, including chromatography on an N-(5'-carboxypentyl)-1-deoxynojirimycin column, resulted in highly purified enzyme with no detectable contamination by other alpha- and beta-aryl-glycosidases. The purification procedure reproducibly yielded 40 microg of pure enzyme per gram wet biomass. Enzyme that was purified using an alternative procedure contained minor impurities and was hydrolyzed by an endogenous proteolytic activity to peptides that retained full catalytic activity. Controlled trypsin hydrolysis of the highly purified enzyme released polypeptide(s) containing the alpha-glucosidase I catalytic domain, with no loss of catalytic activity. This suggests that the catalytic domain of yeast alpha-glucosidase I is resistant to trypsin hydrolysis and remains fully functional after cleavage.
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PMID:An improved purification procedure for soluble processing alpha-glucosidase I from Saccharomyces cerevisiae overexpressing CWH41. 1468 Sep 56

An alpha-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-alpha-D-glucopyranoside (MUalphaGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 degrees C. Although the enzyme preferentially hydrolysed nigerose (alpha1,3-linked), it also cleaved kojibiose (alpha1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (alpha1,4), trehalose (alpha1,1) and isomaltose (alpha1,6). Activity on alpha1,3- and alpha1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy-D-glucose and 6-deoxy-D-glucose were strong inhibitors of activity, whereas 2-deoxy-D-glucose and other monosaccharides had no effect. Enzyme activity on MUalphaGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)-N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.
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PMID:Purification and biochemical characterisation of a membrane-bound alpha-glucosidase from the parasite Entamoeba histolytica. 1501 35

The sea urchin embryo is a model for studying cellular interactions that occur in higher organisms because of its availability, transparency, and accessibility to molecular probes. In previous studies, we found that the mannose/glucose-binding lectin Lens culinaris agglutinin entered living sea urchin embryos, bound to specific cell types and caused exogastrulation, when the developing gut (archenteron) falls out of the embryo proper. We have proposed that the lectin bound to sugar-containing ligands, thus preventing attachment of the archenteron to the blastocoel roof, resulting in exogastrulation. Here, we have continued our study of cellular interactions in this model using Lytechinus pictus sea urchin embryos, and have found that inhibitors of glycoprotein/proteoglycan synthesis, tunicamycin and sodium selenate, and the specific glycosidases, beta-amylase, alpha-glucosidase, and alpha-mannosidase, all inhibit archenteron organization, elongation, and attachment to the blastocoel roof in viable swimming embryos. We also show that single cells obtained by disaggregation of 32-h-old sea urchin embryos bind to L. culinaris agglutinin- and concanavalin A-derivatized beads; the binding is blocked by alpha-methyl mannose, but not l-fucose. These cells also bind to beads derivatized with mannan. These results provide evidence for a role of carbohydrate-containing molecules in cellular interactions in sea urchin gastrulation. In a second set of experiments, we found that the supernatant obtained by disaggregation of 24-32-h-old L. pictus embryos in calcium- and magnesium-free sea water contains molecules that cause exogastrulation, archenteron disorganization, inhibition of archenteron elongation and inhibition of archenteron attachment to the blastocoel roof in viable swimming embryos. We propose that the supernatant contains ligands and/or receptors that mediate archenteron development and attachment to the blastocoel roof and are released when embryos are disaggregated into single cells. These studies may lead to a better understanding of the molecular basis of mechanisms that control cellular interactions during development.
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PMID:Carbohydrate involvement in cellular interactions in sea urchin gastrulation. 1514 30

N-glycosylation inhibitors have antiviral effect against bovine viral diarrhea virus. This effect is associated with inhibition of the productive folding pathway of E1 and E2 envelope glycoproteins. E(rns) is the third pestivirus envelope protein, essential for virus infectivity. The protein is heavily glycosylated, its N-linked glycans counting for half of the apparent molecular weight. In this report we address the importance of N-glycan trimming in the biosynthesis, folding, and intracellular trafficking of E(rns). We show that E(rns) folding is not assisted by calnexin and calreticulin; however, the protein strongly interacts with BiP. Consistently, the N-glycan trimming is not a prerequisite for either the acquirement of the E(rns) native conformation, as it retains the RNase enzymatic activity in the presence of alpha-glucosidase inhibitors, or for dimerization. However, E(rns) secretion into the medium is severely impaired suggesting a role for N-glycosylation in the transport of the glycoprotein through the secretory pathway.
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PMID:Role of N-glycan trimming in the folding and secretion of the pestivirus protein E(rns). 1517 56

A few alpha-L-fucosidase inhibitors and alpha-D-glucosidase inhibitors have shown in vitro anti-HIV activities, that have been attributed to their ability to inhibit HIV entry. The mechanism of action of inhibitors such as 1-deoxynojirimycin (1) is not clearly established. One possible hypothesis is that the glycosidase inhibition affects the final conformation of the glycoproteins involved in the virus/cell recognition and fusion phenomena. This hypothesis is presented critically and the mechanisms of some glycoprotein biosynthesis are out-lined. Up to now, very few glycosidase inhibitors have been assayed for their potential as HIV entry inhibitors. Further assaying should be done and larger collections of glycosidase inhibitors should be prepared. To help investigations in that perspective, the inhibitory activities of alpha-glucosidase and alpha-L-fucosidase inhibitors have been summarized.
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PMID:Glycosidase inhibitors as potential HIV entry inhibitors? 1532 Jul 5


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